Enhanced immunogenicity of tumor associated antigens by addition of alphagal epitopes

ABSTRACT

The invention relates to methods and compositions for causing the selective targeting and killing of tumor cells. The present invention describes prophylactic or therapeutic cancer vaccines based on purified TAA proteins or TAA-derived synthetic peptides altered by chemical, enzymatic or chemo-enzymatic methods to introduce αGal epitopes or αGal glycomimetic epitopes, in order to allow for enhanced opsonization of the antigen by natural anti-αGal antibodies to stimulate TAA capture and presentation, thereby inducing a humoral and cellular immune response to the TAA expressed by a tumor. The animal&#39;s immune system thus is stimulated to produce tumor specific cytotoxic cells and antibodies which will attack and kill tumor cells present in the animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/173,692, filed Jun. 30, 2011, which is a continuation of U.S.application Ser. No. 11/977,203 filed Oct. 24, 2007, now U.S. Pat. No.7,998,476 which claims priority under 35 U.S.C. §120 to provisionalapplication Ser. No. 60/862,840 filed Oct. 25, 2006, each of which isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION Description of the Text File SubmittedElectronically

The contents of the text file submitted electronically herewith areincorporated by reference in their entirety. A computer readable formatcopy of the Sequence Listing (filename: NEWL_(—)012_(—)04US_SeqList.txt,date recorded Dec. 10, 2012, file size 22 kilobytes)

The present invention relates to methods and compositions for treatingcancer by stimulating humoral and cellular immune responses againsttumor cells. In particular, this invention is directed to methods andcompositions to enhance the humoral and cellular immunogenicity ofpurified tumor specific antigens.

BACKGROUND OF THE INVENTION

Despite multiple preventive and therapeutic approaches, cancer is one ofthe major causes of death worldwide. In addition to chemotherapy andradiotherapy, manipulation of the immune system in different types ofimmunotherapies has shown encouraging results in human clinical trials(Berzofsky et al. 2004; Gattinoni et al. 2006). However, newimmunotherapies are greatly needed because currently availabletreatments are still partially effective in cancer eradication(Rosenberg et al. 2004).

Different modalities of cancer vaccines have shown some degree ofclinical efficacy. Whole tumor cell vaccines, administered in presenceof adjuvant and genetically engineered tumor cells that expresscytokines (i.e., granulocyte macrophage colony-stimulating factor andinterleukin 2), are being studied extensively (Dranoff 2002; Rossi etal. 2005a). These vaccines have the advantage of expressing relevanttumor-associated antigens shared by the patient's cancer cells. However,one of the disadvantages of these vaccines is the weak antigenpresentation, poor ability to stimulate a potent immune response and thepotential to cause autoimmune reaction due to non-tumor specificstimulation of immunity against self antigens co-expressed by normalcells (Chianese-Bullock et al. 2005).

The use of complex mixtures of whole tumor cells or tumor-derivedmaterial does not take advantage of the specificity of treatment thatimmunotherapy can provide over other forms of therapy. Theoretically, anantibody or T-cell mediated immune response can recognize uniqueepitopes that are differentially expressed by tumor cells and destroythose cells that express that antigen without affecting normal cells andwithout the risk of triggering an autoimmune response. To take advantageof specificity, large efforts have been invested in the discovery oftumor associated antigens (TAA). Antigen-specific immunotherapyrepresents an attractive approach for cancer treatment because of thecapacity to eradicate systemic tumors at multiple sites in the bodywhile retaining the exquisite specificity to discriminate betweenneoplastic and non-neoplastic cells.

An extensive list of TAAs is available (Novellino et al. 2005; Renkvistet al. 2001). Depending on the specificity of tissue expression andubiquitousness, tumor antigens can be classified in severalcategories: 1) antigens that are expressed only in an individualpatient's tumor; 2) antigens that are commonly expressed in a group oftumors of similar histology; 3) tissue-differentiation antigens; and 4)antigens that are ubiquitously expressed by normal and malignant cellsbut that mutate in tumor cells. Depending on the group of TAA beingtargeted by immunotherapy, the patient treatment will be individualized(group 1) or generic for different patients.

Discovery of such TAAs has prompted many immunotherapy and vaccinationstudies in animals and clinical trials (Antonia et al. 2004; Phan et al.2003; Rosenberg et al. 1998a; Rosenberg et al. 1998b). The firstattempts of immunotherapy using purified TAA proteins with or withoutadjuvant produced disappointing results. For example, immunization ofmice with purified protein of a TAA from syngeneic origin (mouseTyrosinase gp75) does not result in any detectable antibody or T cellresponse to the TAA, due to a pre-established immune tolerance to theunaltered tumor antigen which is also expressed by normal cells(Naftzger et al. 1996). However, immunization of mice with an alteredform of the protein, either by a xenogeneic human gp75 protein or aglycosylated variant of gp75 purified from insect cells was able tobreak the tolerance and induce an antibody response against gp75, thatwas correlated with protection against tumors expressing gp75, and alsoinduction of autoimmunity against normal melanocytes that also expressgp75 (Naftzger et al. 1996). A wide body of evidence supports the notionthat a pre-existing state of tolerance against a self-antigen present intumor cells can be broken by presentation of mutated antigens, antigensin different conformations or with different post-translationalmodifications.

Peptide vaccines composed of short peptides are easier to manufacture inlarge scale than purified protein subunits. Peptide vaccines have beendeveloped by mapping the epitopes from a TAA that bind to the MHCmolecule and that are recognized by the T cell receptor complex. Theseepitopes are 7-13 amino acid sequences derived from the TAA byproteolytic degradation of the TAA in the 26S proteasome. Differentantigenic peptides derived from the same TAA can bind to differenthaplotypes and classes of MHC molecules with different affinities,thereby providing an additional level in the control of specificity ofthe immune response. This means that individual 7-13 amino acid peptidesmight be useful only in patients with appropriate HLA molecules capableof presenting that peptide. Several strategies have been developed toimprove immunogenicity of peptides. Modification of the amino acidsequence of epitopes can improve the efficacy of vaccines by: 1)increasing affinity of peptide for MHC molecules (Berzofsky 1993;Berzofsky et al. 2001; Rosenberg et al. 1998a); 2) increasing binding tothe TCR (Fong et al. 2001; Rivoltini et al. 1999; Zaremba et al. 1997);or 3) inhibiting proteolysis of the peptide by serum peptidases(Berzofsky et al. 2001; Parmiani et al. 2002). Epitope enhancement hasshown greater efficacy in clinical trials (Rosenberg et al. 1998a).However, epitope enhancement is a laborious process that is specific foreach epitope/MHC pair that is being evaluated. Results indicate thatvaccination with TAA-derived peptides can elicit tumor-specific immunityand establish long-term memory without autoimmunity (Scanlan et al.2002; Soares et al. 2001). For example, for breast cancer, vaccinescomposed of epitopes that are derived from melanoma-associated antigen 3(MAGE3) or other members of the MAGE gene family, HER2/NEU (Disis et al.2002), carcinoembryonic antigen (CEA) (Cole et al. 1996; Schlom et al.1996) or mucin 1 have been extensively studied and shown to beimmunogenic without causing autoimmunity. Similarly, for melanoma, manystudies have been undertaken in animal models and in clinical trials(Phan et al. 2003). As these antigens are commonly expressed by tumorsin different patients, large scale production of vaccines can bedeveloped for use in a large number of patients. Despite the advantagesof peptide vaccines and some encouraging preliminary data in animalmodels and clinical trials, tumor vaccines based on individual peptidesderived from TAAs have not produced the results that were initiallyhoped, and often require combinations with potent adjuvants andstimulating cytokines. One of the possible causes of the poorimmunogenic effect of isolated peptides are poor uptake by

APCs, poor activation of APCs by the vaccinating peptides, poor loadinginto the MHC-I and/or MHC-II molecules, poor affinity for certaincombinations of peptide/MHC- specific alleles, pre-existing immunetolerance to self antigens or a combination thereof.

An alternative way to enhance presentation of antigenic epitopes is byuse of in vitro loaded dendritic cells. Mature dendritic cells are themost efficient antigen presenting cells and are the preferred cellulartarget to mediate the elicitation of a potent immune response. For thatreason they have been tested in clinical settings as vaccination vectors(Morisaki et al. 2003). Dendritic cell vaccines are obtained by in vitrodifferentiation of autologous patient-derived CD34+ bone marrow cellswith m-3, IL-6, SCF, GM-CSF, IL-4 or from circulating monocytes byincubation with GM-CSF, IL-4. Immature DCs can be matured in vitro withCD4OL, TNFa or LPS. In vitro differentiated DCs are pulsed with tumorantigen peptides, proteins or tumor cell lysates. Some immunological andclinical responses have been reported for melanoma, follicular B celllymphoma, multiple myeloma and pancreatic cancer, but results have notbeen completely satisfactory, possibly due to inconsistencies in DCspreparation and pulsing (Berzofsky et al. 2004). Therefore, there isstill much room for improvement. The main disadvantage of this approachis that it constitutes a personalized therapy specific for each patient,which limits the scalability of the procedure. DCs need to be collectedfrom each patient, cultured and differentiated in vitro, which is acostly and labor intensive procedure. The inconsistencies in the DCsmethods of collection, differentiation, maturation and pulsing can bepotentially overcome by vaccination methods that induce migration andmaturation of immature DCs in vivo. Vaccination with purified antigensin the form of soluble peptides or proteins results in uptake of theseantigens by pinocytosis, endocytocis or phagocytosis through theendosomal-lysosomal pathway, which ultimately delivers peptide ontosurface MHC class II but not to MHC class I complexes. Thereby,vaccination with soluble proteins or peptides in their native form doesresult mainly in a CD4+ mediated immune response but not in a potentstimulation of CD8+ T cells, which is believed to be the main T celltype needed for an efficient immune response against tumors. It has beendemonstrated that uptake of antigen-antibody immunocomplexes by theFcγRI and FcγRIII receptors in DCs mediates activation and maturation ofDCs and promotes cross-presentation of antigen in the context of bothMEC class I and class II complexes, thereby stimulating both CD4+ andCD8+ cells (Ackerman et al. 2005; Heath et al. 2004; Heath and Carbone2001; Palliser et al. 2005; Rafiq et al. 2002; Schnurr et al. 2005).Consistently with this, vaccination of mice with DCs loaded withimmunocomplexes elicits a protective antitumor response against tumorsbearing the antigen present in the immunocomplex (Rafiq et al. 2002). Itis important to highlight, however, that in this study the animals didnot have a pre-existing state of immunotolerance against the vaccinatingantigen.

An efficient way to promote the formation of immunocomplexes in vivo isby modifying the antigen to contain epitopes or mimotopes against whichthe recipient host has naturally occurring pre-existing antibodies. Thiscan be accomplished by several means such as by introducing A or B bloodantigen groups and administering the modified antigen to an O-type bloodrecipient. Alternatively, a preferred method is to modify the antigen tocontain αGal epitopes (Galα1-3)Galβ(1,4)GlcNAc-R) that would berecognized by natural anti-αGal antibodies existing in humans. Theformation of immunocomplexes by anti-αGal antibodies and αGal epitopeswas first observed during organ xenotransplantation. When transplantingan organ from a non-primate mammal into an Old World primate, the organis destroyed by a hyperacute reaction within minutes of transplantation(Joziasse and Oriol 1999; Maruyama et al. 1999). The hyperacuterejection of xenotransplants to higher primates is mediated by thebinding of anti-αGal antibodies from the recipient to αGal epitopesexpressed on the xenograft and complement activation through the classicpathway (Joziasse and Oriol 1999). In addition, noncomplement fixingnatural anti-αGal antibody induces antibody dependent cell-mediatedcytotoxicity (ADCC) that initiates tissue damage in xenotransplantsmediated by natural killer cells (Baumann et al. 2004; Schaapherder etal. 1994; Watier et al. 1996a; Watier et al. 1996b). The gene encodingfor α(1,3)-galactosyltransferase (αGT), which catalyzes the synthesis ofαGal epitopes on glycoproteins and glycolipids, is inactive in humansand Old World primates but is functional in other mammals (Larsen et al.1990). The human immune system is continuously stimulated by intestinaland pulmonary bacterial flora to produce natural antibodies thatrecognize αGal epitopes. Anti-αGal constitutes approximately 1% ofcirculating IgG (Galili et al. 1984; Galili et al. 1988) and is alsofound in the form of IgA and IgM (Davin et al. 1987; Sandrin et al.1993). It is produced by 1% of circulating B lymphocytes (Galili et al.1993).

It has been demonstrated that immunogenicity of viral or xenogeneicproteins, against which there is no pre-established tolerance, isenhanced by introduction of αGal epitopes. For example, immunization ofαGT knockout mice with BSA conjugated with αGal led to significantproduction of anti-BSA IgG antibodies without the need for adjuvant. Thepresence of αGal also led to an increase in the T cell response to BSA(Benatuil et al. 2005). Additionally, it has been shown that thepresence of anti-αGal antibodies enhanced the cytotoxic T cell responseagainst a viral antigen following vaccination with MoMLV transformedcell lines that express αGal on their surface (Benatuil et al. 2005).Similarly, enzymatic modification of influenza hemagglutinin withrecombinant αGT results in addition of αGT epitopes to HA. It has beenshown that αGal⁽⁺⁾ HA present in whole virions increases the uptake andT cell stimulating capacity of antigen presenting cells, which isreflected by increased proliferation of a HA-specific T cell clone(Galili et al. 1996). Finally, it was recently shown that cxGT KO mice(that were pre-induced to have anti-αGal antibodies) vaccinated withenzymatically modified αGal⁽⁺⁾ HIV-1 gp120 envelope protein induces atleast 100-fold higher titer of anti-gp120 antibodies than micevaccinated with the same dose of an unmodified αGal⁽⁻⁾ gp120(Abdel-Motal et al. 2006). In addition, mice vaccinated with αGal⁽⁺⁾gp120 had higher titer of HIV-1 neutralizing antibodies and largernumber (˜10-fold) of T cells reactive to αGal⁽⁻⁾ gp120. This indicatesthat the presence of αGal epitopes in conjunction with anti-αGalantibodies can provide an adjuvant effect that allows for efficient Tcell and B cell priming to native protein antigens that do not bear αGalepitopes. In these previous experiments, the αGT KO hosts did not have apre-existing state of immune tolerance against the αGal⁽⁺⁾ antigens. Itis not known whether a pre-existing state of tolerance to self antigensor TAA can be broken by vaccination with immunocomplexes composed ofαGal⁽⁺⁾ TAA protein or peptides.

We and others have suggested that the hyperacute rejection of whole cellcancer vaccines expressing αGal epitopes could be exploited as newtherapeutic approach to treat human malignancies (Galili 2004; Galiliand LaTemple 1997; LaTemple and Galili 1999; Link et al. 1998). Thehypothesis that humoral immunity to αGal epitopes may induce anticancerimmunity and bypass or break a pre-existing state of tolerance towardsself antigens shared by normal and tumor cells was tested using theα(1,3)-galactosyltranferase knockout (aGT KO) mouse model (Thall et al.1995). We and others have shown that mice with anti-αGal antibodies areprotected when challenged with αGal-expressing cancer cells, whereas noprotection was observed in mice without anti-αGal antibodies (Posekanyet al. 2004; Unfer et al. 2003). Moreover, the rejection of melanomacells expressing αGal epitopes conferred protection against melanomacells lacking the expression of αGal epitopes. Mice that rejected thefirst challenge with live αGal⁽⁺⁾ B16 cells were protected from a secondrechallenge with αGal⁽⁻⁾ B16 (Rossi et al. 2005a; Rossi et al. 2005b).Moreover, strong CTLs were induced in melanoma protected micerecognizing αGal⁽⁻⁾ B16. In addition, vaccination with B16 melanomacells expressing αGal epitopes prevented tumor development (LaTemple etal. 1999). This data supports the hypothesis that cancer vaccinesexpressing TAAs against which the animal is naturally tolerized canbypass or break that tolerance towards tumor antigens and induce apotent cellular immune response to those TAAs when modified to expressαGal epitopes, and administered to an animal with high titers ofanti-αGal antibodies.

Natural anti-αGal antibodies are of polyclonal nature and synthesized by1% of circulating B cells. They are present in serum and humansecretions and represented by IgM, IgG and IgA classes. The main epitoperecognized by these antibodies is the αGal epitope(Galα1-3Galβ1-4NAcGlc-R) but they can also recognize other carbohydratesof similar structures such as Galα1-3Galβ1-4Glc-R,Galα1-3Galβ1-4NAcGlcβ1-3Galβ1-4Glcβ-R, Galα1-3Glc (melibiose), α-methylgalactoside, Galα1-6Galα1-6Glcβ(1-2)Fru (stachyose),Galα1-3(Fucα1-2)Gal-R (Blood B type epitope), Galα1-3Gal andGalα1-3Gal-R (Galili et al. 1987; Galili et al. 1985; Galili et al.1984). Similarly, non-natural synthetic analogs of the αGal epitope havebeen described to bind anti-αGal antibodies and their use has beenproposed to deplete natural anti-αGal antibodies from human sera inorder to prevent rejection of xenogeneic transplants (Janczuk et al.2002; Wang et al. 1999). Therefore, glycomimetic analogs of the αGalepitope could also be used to promote the in vivo formation ofimmunocomplexes for vaccination purposes.

The above mentioned data suggests that in vivo formation ofimmunocomplexes between TAA purified proteins or TAA-derived peptidesmodified to express αGal or αGal glycomimetic epitopes is a viablealternative for antitumor immunotherapy. The use of purified TAAproteins or moreover, the use of immunogenic synthetic peptides derivedfrom the sequence of TAA modified by chemical, chemoenzymatic orenzymatic addition of αGal epitopes has not been proposed before as atherapeutic alternative. This novel form of tumor vaccination would filla need in the field of tumor immunotherapy providing new therapeuticmethods and compositions that would be highly scalable, reproducible,specific and with enhanced immunogenicity.

SUMMARY OF THE INVENTION

The present invention provides vaccines, compositions and a method ofvaccination with purified TAA proteins or peptides modified by additionof αGal epitopes to trigger the in vivo formation of immunocomplexesbetween αGal⁽⁺⁾-TAA and natural anti-αGal antibodies. Modification ofTAA epitopes with αGal increases their immunogenicity by a mechanismthat relies in enhanced FcγR-mediated capture of TAA-anti-αGalimmunocomplexes by APCs, activation and maturation of DCs, and antigenpresentation in the context of both MHC-I and MIFIC-H molecules, therebyeliciting a humoral and cellular immune response against the unmodifiedαGal″ TAA present in tumor cells.

In one embodiment of the invention, TAA proteins are modified byaddition of αGal epitopes either by expression of the TAA gene in a cellthat naturally expresses an active copy of the αGT gene or by expressionof the TAA in a cell that has been genetically engineered to expressαGT.

In one embodiment, the gene sequence encoding the protein of a TAA thatdoes not normally traffick through the Golgi is modified to include anN-terminal ER/Golgi, secretory or plasma membrane localization signal.

In another embodiment, the gene sequence encoding the TAA protein thatis expressed in an αGal(+) cell is modified to encode an amino acidsequence tag fused to the TAA amino acid sequence in order to facilitateits subsequent purification by affinity chromatography orimmunoprecipitation.

In a preferred embodiment, the purification of αGal⁽⁺⁾ TAA also includesa second affinity purification step by affinity chromatography orimmunoprecipitation with anti-αGal antibodies or 1B4 lectin fromGriffonia simplicifolia.

In an alternative strategy, addition of αGal epitopes to purified TAAproteins is performed in vitro by enzymatic, chemo-enzymatic or chemicalmethods. In another embodiment, αGal epitopes are chemically added invitro to TAA-derived synthetic peptides comprising the followingstructural elements: 1) a sequence of 1-20 amino acids at its aminoterminus that contains the acceptor amino acids for the αGal epitopes,2) a central 7-15 amino acid sequence of a TAA epitope known to elicitan immunogenic CD4+ or CD8+ T cell response, and 3) an optional sequenceof 1-20 amino acids at the C-terminus that contains acceptor amino acidsfor addition of αGal epitopes.

In all previous embodiments, αGal epitopes can also be substituted byglycomimetic epitopes of different chemical structure than αGal thatalso bind to natural anti-αGal antibodies.

In the present invention, the purpose of modification of peptides orproteins with αGal epitopes is to mediate the in vivo formation ofimmunocomplexes with natural anti-αGal antibodies. There is noconceptual limitation in the identity of the TAA and therefore, thesevaccines can be designed using any TAA protein or peptide sequence, aslong as the TAA protein or peptides are expressed by the target tumorand presented in the context of HLA class I molecules.

In summary, it is an object of this invention to develop a therapeuticcancer vaccine by modification of purified TAA proteins or TAA-derivedsynthetic peptides by chemical, enzymatic or chemo-enzymaticmodification to introduce αGal epitopes or αGal glycomimetic epitopes,in order to allow for enhanced antigen opsonization by natural anti-αGalantibodies to stimulate TAA capture and presentation, thereby inducing ahumoral and cellular immune response to the TAA expressed by a tumor.

It is a further object of this invention to provide therapeuticpharmaceutical compositions comprising αGal-modified TAA proteins orαGal-modified TAA synthetic peptides.

It is a further object of the invention to provide vaccines,compositions and methods for treatment of tumors, neoplastic cells orother cells, which grow and evade the cellular and humoral immuneresponse.

Other objects of the invention will become apparent from the descriptionof the invention which follows.

DEFINITIONS

Various terms relating to the vaccines, compositions and methods of thepresent invention are used herein above and also throughout thespecification and claims.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range and include each integer within thedefined range. Amino acids may be referred to herein by either theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly acceptedsingle-letter codes. Unless otherwise provided for, software,electrical, and electronics terms as used herein are as defined in TheNew IEEE Standard Dictionary of Electrical and Electronics Terms (5thedition, 1993). The terms defined below are more fully defined byreference to the specification as a whole.

The term “α-(1,3) Galactosyl Transferase encoding sequence”, or “αGTencoding sequence” or “functional α (1,3)galactosyl transferase” refersto any polynucleotide sequence which encodes a protein that formsα-galactosyl (αGal) epitopes by the following reaction:

Galβ(1,4)GlcNAc-R+UDP-Gal→Galα(1-3)Galβ(1,4)GleNAc-R+UDP

This can include variants, modifications, truncations and the like aswell as enzymes from different animal species known to those of skill inthe art and available in Genbank, other publications or databases whichretain the enzymatic function of the aforementioned reaction.

The term “αGal epitope” refers to any glycosydic structure composed ofat least two monosaccharydes units, the first one being a galactosyl orsubstituted galactosyl residue covalently bond in an a(1-3) bondconformation to a second galactosyl or substituted galactosyl residue,wherein that epitope is recognized by anti-αGal antibodies, includingαGal glycomimetic epitopes.

For glycosidic structures, the terms “αGal glycomimetic variant” or“αGal glycomimetic analogs” or “αGal mimotopes” are defined as anyglycosidic structure, disaccharide, trisaccharide, tetrasaccharide,pentasaccharide or higher order saccharide structure, branched orlinear, substituted or unsubstituted by other chemical groups, that isrecognized in an ELISA by anti-αGal antibodies. For the purpose of thisdefinition, the scope of the specificity of anti-αGal antibodiesencompasses all antibodies that can be purified by affinity in a columncomprising HSA-αGal or BSA-αGal, wherein the αGal epitope bound to HSAor BSA is the Galα1-3Galβ1-4Glc-R trisaccharide plus any linker.

The term “alkyl” as used herein, means a straight or branched chainhydrocarbon containing from 1 to 30 carbon atoms. As used herein, asubstituted alkyl refers to molecules in which carbon atoms in the alkylchain have been replaced by O, N or S and one or more hydrogen groupshave been replaced by hydroxyl, alkyl, amino, carbonyl or sulphydryil.Representative examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Canteen, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “animal” as used herein should be construed to include allanti-αGal synthesizing animals including those which are not yet knownto synthesize anti-αGal. For example, some animals such as those of theavian species, are known not to synthesize αGal epitopes. Due to theunique reciprocal relationship among animals which synthesize eitheranti-αGal or αGal epitopes, it is believed that many animals heretoforeuntested in which αGal epitopes are absent may prove to be anti.-αGalsynthesizing animals. The invention also encompasses these animals.

The term “antibody” includes reference to antigen binding forms ofantibodies (e.g., Fab, F(ab)₂). The term “antibody” frequently refers toa polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof which specifically bind andrecognize an analyte (antigen). However, while various antibodyfragments can be defined in terms of the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments such as single chain Fv, chimeric antibodies (i.e.,comprising constant and variable regions from different species),humanized antibodies (i.e., comprising a complementarity determiningregion (CDR) from a non-human source) and heteroconjugate antibodies(e.g., bispecific antibodies).

The term “anti-αGal” includes any type or subtype of immunoglobulinrecognizing an αGal epitope and/or their glycomimetic variants, of anysubtype such as IgG, IgA, IgE or IgM anti.-αGal antibody. For thepurpose of this definition, the scope of the specificity of anti-αGalantibodies encompasses all antibodies that can be purified by affinityin a chromatography column comprising HSA-αGal or BSA-αGal, wherein theαGal epitope bound to HSA or BSA is the Galα1-3Galβ1-4Glc-Rtrisaccharide.

As used herein, the term “antigen” is meant any biological molecule(proteins, peptides, lipoproteins, glycans, glycoproteins) that iscapable of eliciting an immune response against itself or portionsthereof, including but not limited to, tumor associated antigens andviral, bacterial, parasitic and fungal antigens.

As used herein, the term “antigen presentation” refers to the biologicalmechanism by which macrophages, dendritic cells, B cells and other typesof antigen presenting cells process internal or external antigens intosubfragments of those molecules and present them complexed with class Ior class II major histocompatibility complex or CD1 molecules on thesurface of the cell. This process leads to growth stimulation of othertypes of cells of the immune system (such as CD4+, CD8+, B and NKcells), which are able to specifically recognize those complexes andmediate an immune response against those antigens or cells displayingthose antigens.

The term “chemical” with reference to the addition of an αGal epitopeshall mean that addition of an αGal epitope by means other than the useof the enzyme αGT.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

A “coding sequence” or “coding region” refers to a nucleic acid moleculehaving sequence information necessary to produce a gene product, whenthe sequence is expressed.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences and is intended to be included whenever areference to a specific sequence is made. With respect to particularnucleic acid sequences, conservatively modified variants refer to thosenucleic acids which encode identical or conservatively modified variantsof the amino acid sequences. Because of the degeneracy of the geneticcode, a large number of functionally identical nucleic acids encode anygiven protein. For instance, the codons GCA, GCC, GCG and GCU all encodethe amino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein that encodes a polypeptide also, by reference to thegenetic code, describes every possible silent variation of the nucleicacid. One of ordinary skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine; andUGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide of the presentinvention is implicit in each described polypeptide sequence and iswithin the scope of the present invention. As to amino acid sequences,one of skill will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters, adds or deletes a single amino acid or a small percentageof amino acids in the encoded sequence is a “conservatively modifiedvariant” where the alteration results in the substitution of an aminoacid with a chemically similar amino acid. The following six groups eachcontain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Proteins W.H. Freeman and Company.

We define the “percentage of sequence identity” of two amino acidsequences as the number of identical amino acids shared by these twoamino acid sequences after a pairwise alignment divided by the totallength of the shortest sequence of the pair.

We define the “percentage of sequence similarity” of two amino acidsequences as the number of identical amino acids plus conservative aminoacid substitutions shared by these two sequences after a pairwisealignment, divided by the total length of the shortest sequence of thepair.

By “encoding”, “encodes” or “encoded”, with respect to a specifiednucleic acid, is meant comprising the information for translation intothe specified protein. A nucleic acid encoding a protein may comprisenon-translated sequences (e.g., introns) within translated regions ofthe nucleic acid, or may lack such intervening non-translated sequences(e.g., as in cDNA). The information by which a protein is encoded isspecified by the use of codons. Typically, the amino acid sequence isencoded by the nucleic acid using the “universal” genetic code.

The terms “MHC” (Major Histocompatibility Complex) or “HLA” (HumanLuekocyte Antigen) refer to the histocompatibility antigens of mouse andhuman, respectively. Herein, MHC of HLA are used indistinctly to referto the histocompatibility antigens, without a species restriction, andteachings referring to MHC also apply to HLA and vice versa.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells.

With respect to proteins or peptides, the term “isolated protein (orpeptide)” or “isolated and purified protein (or peptide)” or isolatedTAA protein” is sometimes used herein. This term may refer to a proteinthat has been sufficiently separated from other proteins with which itwould naturally be associated, so as to exist in “substantially pure”form. Alternatively, this term may refer to a protein produced byexpression of an isolated nucleic acid molecule.

With reference to nucleic acid molecules, the term “isolated nucleicacid” is sometimes used. This term, when applied to DNA, refers to a DNAmolecule that is separated from sequences with which it is immediatelycontiguous (in the 5′ and 3′ directions) in the naturally occurringgenome of the organism from which it was derived. For example, the“isolated nucleic acid” may comprise a DNA molecule inserted into avector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a procaryote or eukaryote. An “isolated nucleic acidmolecule” may also comprise a cDNA molecule. With respect to RNAmolecules, the term “isolated nucleic acid” primarily refers to an RNAmolecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from RNA molecules with which it would beassociated in its natural state (i.e., in cells or tissues), such thatit exists in a “substantially pure” form.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). Transfection is usuallyreferred to introduction of DNA by physico-chemical means. Transductionis usually referred to introduction of DNA into a cell mediated by aviral or phage vector.

As used herein, “mimotope” refers to molecular variants of certainepitopes that can mimic the immunologic properties of said epitopes interms of its binding to the same antibodies or being recognized by thesame MHC molecules or T cell receptors.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

The term “nucleic acid construct” or “DNA construct” is sometimes usedto refer to a coding sequence or sequences operably linked toappropriate regulatory sequences and inserted into a vector fortransforming a cell. This term may be used interchangeably with the term“transforming DNA”. Such a nucleic acid construct may contain a codingsequence for a gene product of interest, along with a selectable markergene and/or a reporter gene.

The term “opsonization” of an antigen or a tumor cell is meant bindingof the anti-αGal epitopes present in the antigen or on the surface of atumor cell by anti-αGal antibodies thereby enhancing phagocytosis of theopsonized antigen or tumor cell by macrophages, dendritic cells, B cellsor other types of antigen presenting cells through binding of the Fcportion of the antibodies to the FcγR receptors present on the surfaceof antigen presenting cells.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. This same definition is sometimes applied to the arrangementother transcription control elements (e.g. enhancers) in an expressionvector.

The terms “promoter”, “promoter region” or “promoter sequence” refergenerally to transcriptional regulatory regions of a gene, which may befound at the 5′ or 3′ side of the coding region, or within the codingregion, or within introns. Typically, a promoter is a DNA regulatoryregion capable of binding RNA polymerase in a cell and initiatingtranscription of a downstream (3′ direction) coding sequence. Thetypical 5′ promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence is a transcription initiation site (conveniently defined bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase.

As used herein, “polynucleotide” makes reference to adeoxyribo-polynucleotide

The term “peptide” refers to a polymer of 2-50 amino acids. Peptides canbe derived from proteolytic cleavage of a larger precursor protein byproteases, or can be chemically synthesized using methods of solid phasesynthesis. Synthetic peptides can comprise non-natural amino acids, suchas homoserine or homocysteine to serve as substrates to introducefurther chemical modifications such as chemical linkers or sugarmoieties. In addition, synthetic peptides can include derivatizedglyco-aminoacids to serve as precursors of glycopeptides containing theαGal epitope or its glycomimetic variants.

The terms “protein” or “polypeptide” are used interchangeably herein torefer to a polymer of amino acid residues larger than 50 amino acids.The terms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, the protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide” and “protein” are also inclusive of modificationsincluding, but not limited to, phosphorylation, glycosylation, lipidattachment, sulfation, gamma carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondria]DNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “selectable marker gene” refers to a gene encoding a productthat, when expressed, confers a selectable phenotype such as antibioticresistance on a transformed cell.

A “signal peptide” is a peptide chain (approximately 3-60 amino acidslong that directs the post-translational transport of a protein. Signalpeptides may also be called “targeting signals”, “signal sequences”,“transit peptides”, or “localization signals”. The amino acid sequencesof signal peptides direct proteins (which are synthesized in thecytosol) to different subcellular localizations such as the nucleus,mitochondrial matrix, endoplasmic reticulum (ER) and peroxisome. Somesignal peptides are cleaved from the protein by signal peptidase afterthe proteins are transported. Proteins that contain an ER/Golgilocalization signal traverse through the ER and they can be retained atthe ER, at the Golgi, at the plasma membrane or secreted, depending onadditional localization/retention amino acid sequence signals.

The term “therapeutically effective amount” is meant an amount oftreatment composition sufficient to elicit a measurable decrease in thenumber, quality or replication rate of previously existing tumor cellsas measurable by techniques including but not limited to those describedherein.

The term “tumor cell” refers to a cell which is a component of a tumorin an animal, or a cell which is determined to be destined to become acomponent of a tumor, i.e., a cell which is a component of aprecancerous lesion in an animal, or a cell line established in vitrofrom a primary tumor. Included within this definition are malignantcells of the hematopoietic system which do not form solid tumors such asleukemias, lymphomas and myelomas.

The term “tumor” is defined as one or more tumor cells capable offorming an invasive mass that can progressively displace or destroynormal tissues.

The term “malignant tumor” refers to those tumors formed by tumor cellsthat can develop the property of dissemination beyond their originalsite of occurrence.

The term “Tumor Associated Antigens” or “TAA” refers to any protein orpeptide expressed by tumor cells that is able to elicit an immuneresponse in a subject, either spontaneously or after vaccination. TAAscomprise several classes of antigens: 1) Class I HLA restricted cancertestis antigens which are expressed normally in the testis or in sometumors but not in normal tissues, including but not limited to antigensfrom the MAGE, BAGE, GAGE, NY-ESO and BORIS families; 2) Class I HLArestricted differentiation antigens, including but not limited tomelanocyte differentiation antigens such as MART-1, gp100, PSA,Tyrosinase, TRP-1 and TRP-2; 3) Class I HLA restricted widely expressedantigens, which are antigens expressed both in normal and tumor tissuethough at different levels or altered translation products, includingbut not limited to CEA, HER2/neu, hTERT, MUC1, MUC2 and WT1; 4) Class IHLA restricted tumor specific antigens which are unique antigens thatarise from mutations of normal genes including but not limited toβ-catenin, α-fetoprotein, MUM, RAGE, SART, etc; 5) Class II HLArestricted antigens, which are antigens from the previous classes thatare able to stimulate CD4+ T cell responses, including but not limitedto member of the families of melanocyte differentiation antigens such asgp100, MAGE, MART, MUC, NY-ESO, PSA, Tyrosinase; and 6) Fusion proteins,which are proteins created by chromosomal rearrangements such asdeletions, translocations, inversions or duplications that result in anew protein expressed exclusively by the tumor cells, such as Bcr-Abl.

The term “TAA-derived peptides” refer to amino acid sequences of 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids thatbind to MHC (or HLA) class I or class II molecules, and that have atleast 70% amino acid identity sequence with an amino acid sequencecontained within the corresponding TAA. Peptide sequences which havebeen optimized for enhanced binding to certain allelic variants of MHCclass I or class II are also included within this class of peptides. Inone embodiment, the TAA peptides further comprise at least one or moreαGal acceptor amino acids and/or an affinity purification tag. Inanother embodiment, αGal acceptor amino acids flank the TAA peptide.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A cell has been “transformed”, “transfected” or “transduced” byexogenous or heterologous DNA when such DNA has been introduced insidethe cell. The transforming DNA may or may not be integrated (covalentlylinked) into the genome of the cell. In prokaryotes, yeast, andmammalian cells for example, the transforming DNA may be maintained onan episomal element such as a plasmid. With respect to eukaryotic cells,a stably transformed cell is one in which the transforming DNA hasbecome integrated into a chromosome so that it is inherited by daughtercells through chromosome replication. This stability is demonstrated bythe ability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

As used herein, “vaccine” refers to any antigenic composition used toelicit an immune response. The antigenic composition can be unmodifiedpeptides, glycosylated peptides, purified or recombinant proteins orwhole cells or cell fractions. A vaccine can be used therapeutically toameliorate the symptomps of a disease, or prophylactically, to preventthe onset of a disease.

The term “treat” or “treating” with respect to tumor cells refers tostopping the progression of said cells, slowing down growth, inducingregression, or amelioration of symptoms associated with the presence ofsaid cells.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus towhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “xenogeneic” refers to a cell or protein that derives from adifferent animal species than the animal species that becomes therecipient animal host in a transplantation or vaccination procedure.

The term “allogeneic” refers to a cell or protein that is of the sameanimal species but genetically different in one or more genetic loci asthe animal that becomes the “recipient host”. This usually applies tocells transplanted from one animal to another non-identical animal ofthe same species, or to vaccination of an animal with a protein orantigen from a different strain which may contain differences in theamino acid sequence or post-translational modifications.

The term “syngeneic” refers to a cell or protein which is of the sameanimal species and has the same genetic or amino acid sequencecomposition for most genotypic and phenotypic markers as the animal whobecomes the recipient host of that cell line in a transplantation orvaccination procedure. This usually applies to cells transplanted fromidentical twins or may be applied to cells transplanted between highlyinbred animals.

DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic of the structure of lentiviral vector pHSPA,driving the expression of murine αGT under the control of the human PGKpromoter.

FIG. 1B shows the generation of αGal⁽⁺⁾ B16 cells. αGal⁽⁻⁾ B16 cellswere transduced with vector HSPA, stained with IB4 lectin and sorted byFACS. FAGS histograms show the staining of αGal⁽⁻⁾ B16 (top panel) andsorted αGal⁽⁺⁾ B16 (lower panel) stained with FITC-labeled IB4 lectin.

FIGS. 2A-D show the expression of a fusion protein consisting of: asubcellular localization signal, a protein that normally does nottraffick through the ERJGolgi, in this case AcGFP was used as a modelTAA to allow for easy subcellular localization, and the murine sequenceof αGT with a deletion in its own subcellular ERJGolgi localizationsignal. The fusion proteins are cloned in MoMLV retroviral vectors andtransduced into an αGT(−) human cell line A375. Analysis of αGTexpression was carried by staining with IB4 lectin and detected by FAGS.Analysis of AcGFP expression and its subcellular localization wasdetermined by fluorescence microscopy. See Example 8.

FIG. 3 is a schematic description of synthesis of αGal epitopeNLG-αGal-001. See Example 22 for details.

FIG. 4 is a schematic description of synthesis of αGal epitopeNLG-αGal-002. See Examples 22-26 for details.

FIG. 5 is a schematic description of synthesis of αGal epitopeNLG-αGal-003. See Examples 27-29 for details.

FIG. 6 is a schematic description of synthesis of αGal epitopeNLG-αGal-004. See Examples 30-32 for details.

FIG. 7 is a schematic description of synthesis of4-[(13-D-lactopyranosyl) (methyl)aminooxy]-4-oxobutanoic acid. SeeExamples 33 for details.

DETAILED DESCRIPTION OF THE INVENTION

Tumor cells express antigens that can be recognized by the host's immunesystem. Endogenous TAA are degraded in the proteasome into 8-11 aminoacid peptides which bind to the MHC class I. Each allelic MCH variantbinds only a subset of peptides that share conserved amino acid residuesat each position. The peptide-MHC complex is recognized by the T cellreceptor (TCR) on the surface of T lymphocytes. Therefore, an exquisitelevel of specificity is achieved by presentation of certain peptides inthe context of specific MHC classes and allelic variants that arerecognized only by certain TCR molecules.

The basic rationale for immune therapy against tumors is the inductionof an effective and specific immune response against tumor-associatedantigens (TAA), which in turn results in immune-mediated destruction ofproliferating tumor cells expressing these antigens.

Effective prophylactic or therapeutic vaccines based on TAA proteins orpeptides have several requirements. First, the epitopes present in thevaccine have to be present in TAAs expressed by the tumor. Second, theepitopes have to be effectively presented in the context of the rightMHC alleles of the patient. Third, the vaccinating antigens must beproperly captured, processed and presented by antigen presenting cells(APC) such as macrophages, dendritic cells and B cells. Within APCs,TAAs are degraded in the lysosomal compartment and the resultingpeptides are expressed on the surface of the APC membrane mostly inassociation with MI-IC Class II molecules and also in association withMHC class I molecules if the antigen traversed the cross-presentationpathway. This expression mediates recognition by specific CD4+ helper Tcells or CD8+ effector T cells and subsequent activation of these cellsto effect the immune response (Lanzavecchia 1993; Pardoll 1993).

Great challenges have to be overcome to achieve an effectiveimmunization using TAA proteins or peptides. Most tumor cells haveunique expression profiles of TAA, and in many cases the immunogenicpeptides include a mutated amino acid sequence that confersimmunogenicity through the exposure of an altered nonself epitope. Theseepitopes are usually very immunogenic. However, many tumors escapeimmune surveillance either 1) by not-generating these epitopes duringproteasome processing, or 2) by down regulating the expression of MHCcomponents such as β-microglobulin, or 3) because the immune system doesnot recognize these TAA as foreign antigens because either they are notpresented in the context of a cellular “danger” signal, or 4) becausethe immune system has been tolerized to those antigens and recognizesthem as “self” antigens. Immunotherapeutic approaches based on T-cellrecognition of TAA-derived peptides are not expected to work using anyvaccination approach for the two first cases (i.e. when the tumor doesnot present the antigenic TAA), but are well suited for the last twocases (i.e. when the immune system does not recognize the TAA asimmunogenic).

One of the reasons for the lack of a sufficient immune response tocontrol cancer growth in vivo is due to the poor immunogenicity ofnatural epitopes expressed by tumor cells. With the exception of theimmunodominant melanoma Melan-A/MART1₂₇₋₃₅ and gp100 peptides, whichreadily activate specific T cells in vitro (Rivoltini et al. 1995) andin vivo (Cormier et al. 1997), most T-cell responses require repeated invitro stimulation with TAA epitopes and show limited immunogenicity whenused as vaccines for cancer patients (Marchand et al. 1999; Weber et al.1999). New strategies for increasing in vivo immunogenicity consist ofmodifying the peptide sequence at amino acid residues to: 1) improve theinteraction with the HLA or with the specific TCR, 2) inhibitdimerization, 3) reduce or inhibit proteolytic degradation (Brinckerhoffet al. 1999; Chen et al. 2000; Parkhurst et al. 1996; Tourdot et al.2000). In addition, caution should be taken when designing vaccinesbased on peptide immunization as under certain circumstances,vaccination with peptides may induce epitope specific T-celltolerization rather than activation, depending on the adjuvant used androute of immunization.

One possible explanation for the limited therapeutic efficacy of TAApeptide vaccination lies in the fact that activation of peptide-specificCTL responses requires the delivery of inflammatory signals frommonocytes, lymphocytes, or granulocytes recruited at the site ofvaccination. Those signals may or may not be provided by standardadjuvants like incomplete Freund's adjuvant. An efficient activationsignal, however, may be provided by natural adjuvants that trigger a“danger” signal such as bacterial DNA or synthetic oligodeoxynucleotides(ODN) containing unmethylated CpG dinucleotides (CpG-ODN). Such signalscan stimulate B cells, natural killer (NK) cells, T cells, monocytes,and antigen-presenting cells; more importantly, such signals can promotematuration of DCs, a step that will result in the activation of theantibody and cell-mediated immune responses (Brunner et al. 2000;Sparwasser et al. 1998). More recently, CpG ODN have been shown toimprove the antitumor activity of antigen-presenting cells loaded withTAA peptides and promote a 10-fold to 100-fold increase in the inductionof CTL responses to peptide immunization (Brunner et al. 2000).

Another possible reason of poor results obtained through peptidevaccination is the exclusive focusing on peptide epitopes that bind toMHC class I molecules to trigger CTL immune response. Peptide epitopesthat trigger a CD4+ T cell response also result in stimulation andactivation of B cells, leading to a humoral response against TAA.

There are several theoretical reasons that justify why a strong humoralimmune response should produce a more effective anti-tumor response. Infact, passive administration of antibodies against gp75 preventsdevelopment of melanoma metastases in a mouse model (Clynes et al.1998). The binding of antibodies to TAA promotes the formation ofimmunocomplexes, which bind to the FcγR receptors on APCs. Fc receptortargeting accomplishes several important functions for effective vaccineperformance including promoting the efficient uptake of antigen for bothMEC Class I and II antigenic presentation; promoting APC activation andmaturation of dendritic cells. APCs that ingest a tumor cell must beactivated before they can effectively present antigen. Otherwise,presenting antigens to immature APCs, without the required activationsignals, can suppress the immune response. Second, the uptake ofopsonized TAA, or TAA-expressing cells by antigen presenting cells viaFcγR receptor mediated endocytosis may be critical to generating aneffective anti-tumor CTL response since it promotes the activation ofMHC class I restricted responses by CD8+ T-cells through a crosspresentation pathway. Third, vaccines that cannot stimulate a humoralimmune response are limited in their ability to induce cellular immunityby HLA restriction. CTLs are HLA restricted and will only destroy thetumor cells that present TAAs on self-class I MHC molecules. On thecontrary NK cells will destroy the tumor vaccine cells if they areopsonized by antibodies by antibody-dependent cell cytotoxicity (ADCC).

The present invention provides methods and composition for peptidevaccines that contemplate the aspects mentioned above and overcomes someof the current limitations associated with the development of TAAprotein or peptide vaccines. In the present invention, TAA proteins orTAA-derived peptides are modified by functionalization with a αGalepitope which promotes the in vivo formation of immunocomplexes withnatural anti-αGal antibodies. This provides several immunologicadvantages over the use of other vaccines composed of TAA proteins orpeptides (with or without adjuvant), which do not promote the in vivoformation of immunocomplexes.

First, the binding of natural anti-αGal IgG or IgM to αGal epitopespresent in the immunizing TAA molecule facilitates the formation ofimmunocomplexes, which triggers complement activation and opsonizationof the immunocomplex by C3b and C3d molecules, which can target theimmunocomplex to follicular dendritic cells and B cells via CD21 andCD35, thereby augmenting the immune response. Also, FcγR receptormediated phagocytosis of IgG immunocomplexes by DCs is a very efficientmechanism of antigen uptake and processing. Second,complement-activation at the site of vaccination generates a “dangersignal” which has numerous implications for the kind of immune responsethat will be generated (Matzinger 2002; Perez-Diez et al. 2002). Dangersignals are recognized as crucial components for APC activation anddifferentiation to mature DCs. Additionally, complement activation haschemo-attractant properties that, similarly to GM-CSF, result ininflammation and recruitment of APCs.

Different antigen uptake and processing pathways control thepresentation of antigenic peptides by either MHC class I molecules toCD8+ T cells (endogenous pathway) or MHC class II molecules to CD4+ Tcells (exogenous pathway). Vaccines that are composed of exogenousantigens use mainly the exogenous pathway for the delivery of antigen toAPCs. This, in turn, favors the stimulation of CD4+ T cells and theproduction of antibodies. To deliver exogenous antigens to theendogenous pathway in order to elicit a cellular mediated response, theengagement of the FcγR receptor to mediate antigen uptake ofimmunocomplexes is very important as it stimulates thecross-presentation pathway (Heath and Carbone 2001). Studies indicatethat, in addition to classical CD4+ priming, antigen acquired throughendocytosis by DC through FcγR results in the induction of T celleffector immunity resulting in T_(H)1 and class I restricted CD8+ T cellpriming. Furthermore, engagement of FcγR also induces DC activation andmaturation. Thus, the existing evidence indicates that antigenictargeting to FcγR on DC accomplishes several important aspects of T cellpriming important for induction of an immune response: facilitateduptake of antigen, class I and class II antigen presentation andinduction of DC activation and maturation.

In the specific case of αGal⁽⁺⁾ TAA vaccines of the present invention,three mechanisms of antigen uptake are expected to take place. First,the exogenous pathway involving phagocytosis/pinocytosis that sends theantigens through the endosomal/lysosomal pathway which results inpresentation of the processed antigen in the context of MI-1C class IIsurface molecules that activate the proliferation of CD4⁺ helper Tcells. Second, FcγR-mediated antigen uptake of immunocomplexes involvinganti-αGal antibodies will favor the cross-presentation pathway,resulting in antigen presentation in the context of MHC class Imolecules, which will preferentially activate CD8⁺ cytotoxic T cells.Third, binding of tumor specific antigen molecules to membrane IgMpresent in naïve B-cells will result in B-cell activation anddifferentiation, and also in MHC class II antigen presentation thatfurther stimulates proliferation of memory CD4⁺ T-cells that recognizethose antigens. After activation and stimulation B-cells proliferate,differentiate and produce antibodies which bind to surface TAA moleculespresent on the target tumor cells, facilitating killing of the cell bycomplement-mediated cell lysis, antibody dependent cell cytotoxicity andFcγR-dependent phagocytosis. Also, target cell destruction is mainlyachieved by cytotoxic CD8⁺ T cells previously activated bydifferentiated dendritic cells and helper CD4′ T cells. In summary, amain advantage of the αGal⁽⁺⁾ TAA vaccines of the present invention overprevious TAA protein or peptide vaccines is that it achieves the in vivoformation of immunocomplexes in the absence of adjuvant. This leads torecruitment of antigen presenting cells, increased FcγR-mediatedphagocytosis and antigen uptake that result in activation of bothcellular and humoral branches of the immune response. The strongerinitial immune reaction is expected to induce both a more effectiveimmunity and the generation of a larger pool of memory cells. Therefore,taking advantage of the strong innate immune response toa-galactosylated proteins establishes a firm basis for novel antitumorand antiviral immunotherapies.

Theoretically, there is no limitation in the identity or properties ofthe TAA used for vaccination. A vast list of TAA has been compiled byRenkvist et al. (Novellino et al. 2005; Renkvist et al. 2001). All theTAA antigens cited in these publications are suitable for the method andcompositions of the present invention and are incorporated herein byreference. Similarly, portions of the full length TAA amino acidsequences or their isoforms are well suited for the purposes ofantitumor vaccination described in this invention.

Tumors which may be treated in accordance with the present inventioninclude malignant and non-malignant tumors. Malignant (including primaryand metastatic) tumors which may be treated include, but are not limitedto, those occurring in the adrenal glands; bladder; bone; breast;cervix; endocrine glands (including thyroid glands, the pituitary gland,and the pancreas); colon; rectum; heart; hematopoietic tissue; kidney;liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx;larynx; ovaries; penis; prostate; skin (including melanoma); testicles;thymus; and uterus. Examples of such tumors include apudoma, choristoma,branchioma, malignant carcinoid syndrome, carcinoid heart disease,carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal,Ehrlich tumor, in situ, Krebs 2, Merkel cell, mucinous, non-small celllung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic,squamous cell, and transitional cell), plasmacytoma, melanoma,chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giantcell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma,myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma,adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma,mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma,thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma,cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosacell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor,Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor,leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma,rhabdomyosarcoma, ependymoma, ganglioncuroma, glioma, mcdulloblastoma,meningioma, neurilemnnoma, neuroblastoma, neuroepithelioma,neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin,angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angiomasclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma,hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyorna,lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma,cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma,leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma,ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing'sexperimental, Kaposi's, and mast-cell), neoplasms and for other suchcells.

In one embodiment of the invention, TAA proteins are modified byaddition of αGal epitopcs by expression in cultured cells. This can beaccomplished by several means. In a preferred embodiment, recombinantTAA proteins are expressed in eukaryotic cells that naturally express anactive copy of the gene encoding for αGT. These cell lines can bederived from any mammalian species except the catharrinc primates suchas apes, humans or Old World monkeys that do not express αGT. Expressionof αGT and presence of αGal epitopes can be confirmed by staining cellswith Griffonia simplicifolia 1B4 lectin or with purified anti-αGalantibodies and subsequent analysis by FACS. There are numerous examplesin the literature of vector systems for the expression of recombinantproteins in mammalian cells (Schmidt 2004).

In an alternative embodiment, recombinant TAA proteins are expressed ina eukaryotic cell line that has been genetically modified to express thegene encoding a functional αGT enzyme. αGT has been cloned from severalmammalian species such as mouse, bovine, sheep, porcine, cat, dog andmarmoset (Hellion et al. 1994; Joziasse et al. 1989; Joziasse et al.1992; Koike et al. 2002; Strahan et al. 1995a; Strahan et al. 1995b;Taylor et al. 2003). Stable transfectants of mammalian cells can beobtained by stable transfection with plasmid DNA or by stabletransduction with viral vectors encoding αGT under the control of aconstitutive or inducible promoter. Viral vectors suitable for thistransfection comprise the use of retroviral vectors such as Moloneymurine retrovirus or human, simian, feline or equine lend viral vectorsby methods well described in the art (Phillips 2002).

In an alternative embodiment, the TAA protein can be expressed in agenetically engineered yeast cells such as Saccharoinyces cerevisiae orPichia pastoris, modified to express a recombinant αGT, such as the onedescribed by Shao et al. (Shao et al. 2003).

Addition of αGal epitopes to recombinant TAA protein expressed in aeukaryotic cell that also expresses a functional copy of αGT requiresthat the TAA protein trafficks through the Golgi via theexcretory/secretory pathway in order to become glycosylated with αGalepitopes. αGT localizes to the Golgi and catalyzes the transfer ofUDP-Gal to a lactosaminyl or N-acetylylactosaminyl acceptor that is partof N-linked or O-linked oligosaccharide structures. N-Linked glycans arelinked to the protein backbone via an amide bond to asparagine residuesin an Asn-X-Ser/Thr motif, where X can be any amino acid, except Pro.O-Linked glycans are linked to the hydroxyl group of serine orthreonine. Therefore, in order to effect the in vivo addition of αGalepitopes to a recombinant TAA protein, this protein has to satisfy thefollowing requirements: 1) possess an N-terminal ER/Golgi localization,Golgi trafficking, secretory or plasma membrane localization signal, 2)possess a consensus for N-glycosylation or exposed Ser/Thr amino acidsin hydrophilic loops of the protein for O-glycosylation.

In a preferred embodiment, the recombinant TAA encoding gene is modifiedto allow its encoded protein to be glycosylated and secreted through thesecretory pathway, into the cell culture medium, thereby facilitatingextraction and purification of the αGal-modified TAA from the growthmedium. In the case of TAA derived from membrane glycoproteins,expression of the soluble extracellular glycoprotein domain bytruncation of the membrane anchoring domain(s) and inclusion ofsecretory signals would allow to recover the αGal⁽⁺⁾ TAA soluble domainin the cell culture medium. This embodiment would facilitate continuousculture and production of αGal⁽⁺⁾ TAA protein. Alternatively, theαGal⁽⁺⁾ TAA recombinant protein can be purified from lysed cellsfollowing protein purification protocols suitable for each specificprotein.

A few examples of ER/Golgi-localization or secretory signals include thefollowing amino acid sequences and conservative variants thereof:

(SEQ ID NO: 15) 1] MRVLVLALAVALAVGDQSNLG [U.S. Pat. No. 6,733,997](SEQ ID NO: 16) 2] MKWVTFLLLLFISGSAFSR [Preproalbumin] (SEQ ID NO: 17)3] MDMRAPAQIFGFLLLLFPGTRCD [Pre-IgG light chain] (SEQ ID NO: 18) 4]MRSLLILVLCFLPLAALGK [Prelysozyme] (SEQ ID NO: 19) 5]MMSFVSLLLVGILFWATDADNLTKCDVFN (SEQ ID NO: 12) 6]MDLLLLLLLGLRLQLSLGRIP [aGT]

A few examples of Golgi localization sequence signals include thefollowing amino acid sequences and conservative variants thereof:

1] Arg-based signals with the consensus Z/Z/R—R—X—R (SEQ ID NO:82), inwhich Z denotes an aromatic or bulky hydrophobic residue and Xrepresents any amino acid. More than two arginine residues gives rise toparticularly strong sorting motifs, whereas the residue that precedesRxR and the identity of X itself can modulate the signal to anintermediate efficacy that results in significant steady state Golgilocalization

2] C-terminal -K(X)KXX (SEQ ID NO:83) ER-localization signals, exposedat the distal terminus of a membrane protein.

Several methods can be used to purify the αGal⁽⁺⁾ TAA recombinantprotein from cell lysates or from the cell culture medium. A commonapproach is to modify the sequence of the gene encoding the TAA toinclude an affinity tag fused to the TAA protein. Examples of affinitypurification tags are well known in the art and include: 1)polyhistidine tags to facilitate protein purification in a Ni⁺² column;2) glutathion S-transferase; 3) maltose binding protein [New EnglandBiolabs]; 4) chitin binding protein [New England Biolabs]; 5) amino acidsequence tags recognized by immobilized monoclonal antibodies, such asthe V5, HA, FLAG sequences. In a preferred embodiment, such tags areincluded at the C-terminus of the recombinant TAA to avoid interferencewith the secretory sequence signals. In an alternative embodiment, theaffinity purification tag is fused to the TAA recombinant protein via aprotease sensitive sequence to allow separation of the sequence tag fromthe TAA protein.

As a single step of protein affinity purification from a complex proteinmixture generally does not yield a pure protein preparation, a preferredembodiment for protein purification of αGal⁽⁺⁾TAA is to include a secondpurification step based on affinity to the αGal epitope. Natural humananti-αGal antibodies or the IB4 lectin from Griffonia simplicifoliarecognize αGal epitopes on glycoproteins. An affinity matrix columncontaining covalently linked anti-αGal antibodies or 1B4 lectin can beused to purify the αGal⁽⁺⁾ TAA and yield a more pure TAA proteinpreparation.

An alternative approach to addition of αGal epitopes to TAA proteins byexpression in αGal⁽⁺⁾ cells is to perform the addition of αGal epitopesin vitro. In vitro approaches use purified TAA protein or proteinfragments/domains obtained from any cell expression system (bacteria,yeast, mammalian, insect cells), regardless of whether these cellsexpress αGT or not. Subsequent addition of αGal epitopes can beperformed by: 1) enzymatic methods, 2) chemo-enzymatic methods or 3)chemical modification. The preferred method of addition of αGal epitopeswill be chosen depending on the cell expression system used as thesource of recombinant TAA protein.

Enzymatic addition of αGal epitopes to whole cells, membrane fractionsor recombinant proteins purified from mammalian cells has been describedextensively by Galili et al. and those methods are incorporated hereinby reference (Abdel-Motal et al. 2006; Chen et al. 2001; Galili et al.2001; Henion et al. 1997; LaTemple et al. 1996). Briefly, when TAAglycoproteins are purified from αGal⁽⁻⁾ mammalian cells, two enzymaticsteps have to be performed to mediate addition of αGal epitopes. Thefirst step is the removal of the terminal sialic acid residues from theglycosyl structures by incubation with the enzyme neuraminidase. Thesecond step is the incubation of such protein with recombinant purifiedαGT and UDP-Gal to mediate the addition of Galactose in an a(1-3)configuration to N-acetyl-lactosaminyl (Gal β1-4GlcNAc-R) orlactosaminiyl (Gal β1-4Glc-R) acceptor residues. The incubation withneuraminidase can be obviated though the efficiency of incorporation ofαGal epitopes increases ˜5-fold after addition of this enzymatic step(Chen et al. 2001). Also, removal of sialic acid residues withneuraminidase can be performed as a first step or sequentially with αGTincubation. The αGal⁽⁺⁾ TAA has to be purified from this mixture, by anyof the means described above.

For instances in which the TAA protein is purified from cell expressionsystems based on insect or yeast cells, the glycosylation pattern willdiffer from the glycosylation pattern observed in TAA purified frommammalian cells. Insect cells will provide a less complex and moreimmature high-manose core that can be used as an acceptor to performsequential enzymatic addition of 1) UDP-GleNAc by N-acetyl glucosaminyltransferase I, 2) UDP-Gal by a β(1-4) galactosyl transferase and 3)UDP-Gal by αGT.

If the TAA protein or protein domain is purified from bacterialexpression systems, it will not have an appropriate lactosaminylacceptor for UDP-Gal and αGT will not be able to catalyze addition ofotGal epitopes in vitro. Therefore, the chemical addition of activatedαGal cpitopes is the method of choice in this case.

Several methods have been described for the purely chemical, purelyenzymatic or a combination of chemo-enzymatic synthesis of αGal orαGal-derivative epitopes with the purpose to inhibit binding ofanti-αGal antibodies to cells expressing αGal epitopes. For example, thesynthesis of the following αGal epitopes has been previously describedand their methods of synthesis are incorporated by reference herein:

-   -   1] Galα1-3Gal-R—NH₂ (Wang et al. 1999)    -   2] Galα1-3Galβ1:4 Glc-R—NH₂ (Wang et al. 1999)    -   3] Galα1-3Galβ1-4Glc-N₃ (Fang et al. 1998)    -   4] Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-N₃ (Fang et al. 1998)    -   5] Galα1-3Gal-O—(CH₂)₆—NH₂ (Hanessian et al. 2001b)    -   6] Galα1-3Galβ1-4GlcNAc-O—(CH₂)₃—NH₂ (Hanessian et al. 2001b)    -   7] Galα1-3Galβ1-4GlcNAcβ-OBn (Reddy et al. 1994)    -   8] Galα1-3Galβ1-4GlcNAcβ-6GalNAc-a-OBn (Reddy et at 1994)    -   9] Galα1-3Galβ1-4Glc-O—(CH₂)₃—NH₂ (Hanessian et al. 2001a)    -   10] Galα1-3Galβ1-4-R—NH₂ (Hanessian et al. 2001a)    -   11] A trimeric cluster of Comp #10 on penta-erythritol scaffold        containing a free terminal NH₂ group (Hanessian et al. 2001a)    -   12] Galα1-3Galβ1-4Glc-O—(CH₂)₂—O—(CH₂)₂—NH₂ (Naicker et al.        2004)    -   13] Galα1-3Galβ1-4Glc (Shao et al. 2003)    -   14] Galα1-3Galβ1-4Glc-NAc-(CH₂)₃—S—(CH₂)₂—NH₂    -   15] Galα1-3Galβ1-4GlcNAc-SEt

αGal epitope compounds #1, 2, 5, 6, 9, 10, 11 and 12 have a free primaryNH₂ group. αGal epitope compounds #3 and 4 have an azide group that canbe easily converted to a primary amino group by reduction in H₂/Pd—C.Compound #13 is easily produced in large amount from an inexpensivesource of sucrose and lactose by a genetically engineered PichiaPastoris cell line (Shao et al. 2003). This trisaccharide can befunctionalized with allylamine and cysteamine hydrochloride at the1β-anomeric position by a method similar to the one described by Ramoset al. (Ramos et al. 2001) to yield compound #14 which has a primaryamino group.

The primary amino group can be coupled to a bifunctionalN-hydroxy-succinimide-Ll-Maleimide cross-linker (NHS-R-Mal, where LI isany type of linear linker such as but not limited to: alkyl, ether,polyether or polyamide) to yield a reactive αGal epitope. This Maleimideactivated αGal molecules can be reacted to Cysteine residues in thepurified TAA protein, thereby yielding a αGal(+)TAA protein.

Alternatively, αGal epitopes having a primary amino group can beenzymatically coupled to the γ-carboxamide residue of glutamine bybacterial glutaminyl-peptide γ-glutamyl transferase (Transglutaminase)(Ramos et al. 2001). Synthesis of compound #15 by the sequentialenzymatic activity of β-galactosidase (from Bullera singularis orBacillus circulars) and α-galactosidase (from Aspergillus oryzae orgreen coffee beans) from 1-βD-thioethylglucosamine, O-p-nitrophenylα-D-galactopyranoside and O-p-nitrophenyl β-D-galactopyranoside has beenpreviously described (Nilsson 1997; Vic et al. 1997). Reduction ofthioethyl group to sulphydryl group leaves a free —SH group that isreactive with Maleimide-R2-NHS linkers. This activated αGal epitope canbe coupled to proteins or peptides bearing primary amino groups eitherat the N-terminus or at lysine residues. Similarly, bifunctionalNHS—R1-NHS linkers could be coupled to the free NH₂ group of αGalmolecules (such 1, 2, 5, 6, 9, 10, 11 and 12) and then coupled to theε-NH₂ group of lysines present in the TAA protein or peptides.

In a preferred embodiment, the αGal epitopes with the generic structure

αGal-L₁-R₂

are synthesized by the methods of the present invention. LI is a linkerwith the preferred structure —N(CH₃)—OR₁—COO—, wherein R₁ is any linearor branched alkyl group of 1 to 30 carbon atoms, wherein one or morecarbon atoms in such alkyl group can be substituted by O, S, or N andwherein one or more hydrogens can be substituted by hydroxyl, carbonyl,alkyl, sulphydryl or amino groups. In a more preferred embodiment, suchatom substitutions create one or more ester groups situated at anyposition within the R1 alkyl chain. R₂ is any functional group that isreactive to primary amino groups, sulphydryl groups or acid groups, suchas N-hydroxysuccinimide, or maleimide. First, the linker of structureNH(CH₃)—O—R₁—COOH is reacted to synthetic or semisynthetic αGaltrisaccharide, such as the one purified from a genetically engineeredPichia Pastoris described by Shao et al, or from any commercial source.The resulting αGal-tinker molecule is then activated withN-hydroxysuccinimide or maleimide, thereby yielding an activated αGalepitope that can be reacted with any primary amino group present in anylysine or cysteine residue or N-terminal amino group of any protein orpeptide. The methods and compositions described here for the synthesisof αGal-L₁-R₂ activated molecules also apply for any αGal epitope andalso to any monosaccharide, disaccharide, trisaccharide, tetrasaccharideand pentasaccharide. Examples of such activated αGal epitopes arecompounds of the formula:

Details of the synthesis schemes and reaction conditions to obtain suchcompounds are provided in FIGS. 3-6 and Examples 22-32.

The above mentioned αGal epitopes could also be used to modify syntheticpeptides that bear amino acids such as Cysteine, Homocysteine, Serine,Threonine or Glutamine, by post-synthesis chemical conjugation of theactivated αGal epitope to the pure synthetic peptide in the same way asdescribed for TAA proteins.

It is important to highlight the fact that αGal epitopes added by αGTwill be in a different chemical context as the αGal epitopes added bychemical means. Therefore, the αGal⁽⁺⁾-TAA vaccine compositionsgenerated by chemical addition of αGal epitopes will differ from theαGal⁽⁺⁾-TAA vaccine compositions generated by enzymatic addition of αGalepitopes and as such, constitute a novel chemical entity. The differentchemical context to which αGal epitopes are added within a protein orpeptide might affect the reactivity of different species of anti-αGalantibodies present in human serum towards the different classes of αGalepitopes. However, given the vast polyclonality of antibody species thatrecognize the αGal epitopes, the overall efficacy of the vaccine willnot be affected by the chemical context of the αGal addition site.

In the present invention, the purpose of modification of peptides orproteins with αGal epitopes is to mediate the in vivo formation ofimmunocomplexes with natural anti-αGal antibodies, thereby facilitatingFcγR-mediated uptake of the immunocomplex, which will ultimately lead toenhanced presentation of the deglycosylated immunogenic epitopcs,thereby triggering immunity against the native TAA expressed by thetumors which is not modified with αGal. Therefore, some considerationsregarding processing and presentation of glycosylated antigens areimportant to take into account when performing chemical modification ofproteins or peptides. Glyeoprotein antigens are ingested by APCs byendocytosis and transported from the cell surface toward the lysosomalcompartments. During transport, proteolytic enzymes become activated asthe pH of the endosome decreases. The enzymes, which includeendoproteases and exoproteases with many different substratespecificities, attack and fragment the antigen into peptides (Cresswell2005; Cresswell et al. 2005; Kloetzel 2001; Kloetzel 2004; Kloetzel andOssendorp 2004; Kruger et al. 2003; Li et al. 2005; Rock et al. 2004).Glycans in a glycoprotein or glycopeptide can interfere with theproteolytic fragmentation and influence the pattern of T cell epitopesthat are presented. Appropriate peptides (8-15 amino acids) areprotected against further proteolysis as they bind to empty MEC class IImolecules that are accumulating within the acidic compartments. Finally,the MHC-peptide complexes are transported to the cell surface andpresented to CD4+ T cells. Due to the fact that many cellular proteinsare extensively glycosylated, processing and presentation mechanisms areexpected to produce a pool of major MHC-bound protein-derived peptides,part of which retain sugar moieties. It has been demonstrated that Tcells are able to recognize partially glycosylated peptides that bind tothe MHC molecules if the sugar moiety is small and if it is located in acentral position within the peptide being presented (Speir et al. 1999;Werdclin et al. 2002). Sugar moieties present at the ends of the peptidebeing presented do not elicit and immune response against theglycosylated portion of the peptide. In the present invention, theobjective is to elicit an immune response against deglycosylatedpeptides or against the non-glycosylated portion of the glycopeptides,since the target TAAs expressed by tumors do not bear the sameglycosydic modification as the immunizing peptides. Since the chemicaladdition of αGal epitopes mediated by N-hydroxysuccinimide,

-   Maleimide or other functional groups will not create the natural    N-linked chemical bonds of sugar to Asparagine residues, or the    natural O-linked sugar moieties to Serine or Threonine residues,    complete removal of sugar moieties (that do not contain natural    N-linked or O-linked chemical bonds) is anticipated to be impaired    during antigen processing.

In a preferred embodiment, removal of the αGal epitope bound to apeptide or protein during antigen processing and presentation can befacilitated by including one or more ester groups in the linker bridgingthe trissacharide portion of the αGal epitope with the peptide orprotein. After endocytosis, intracellular esterases of differentspecificities cleave the αGal epitope at the ester group present in thelinker region, thereby yielding a deglycosylated peptide that can bindto the MHC class I and H and elicit an immune response by engaging withTCR present in CD4+ and CD8+ T cells.

An alternative embodiment to prevent potential difficulties associatedwith incomplete deglycosylation of immunizing glycopeptides is toseparate the region of the peptide known to trigger an immune responseagainst cells expressing the corresponding TAA from the regionconjugated to the αGal epitope. This can be done by creating a αGal tagfused to the immunogenic peptide. The αGal tag consists of a stretch of1 to 20 amino acids that bear the amino acids to which the αGal epitopewill be covalently linked to, in addition to known endoprotease aminoacid consensus sequences that will facilitate its cleavage by endosomalproteases. In this way, the αGal tag will mediate formation ofimmunocomplex with anti-αGal antibodies, thereby enhancing DCactivation, antigen processing and presentation. The αGal tag will bereleased from the immunogenic portion of the peptide by proteases andaminopeptidases during antigen processing. The release of thenon-glycosylated immunogenic portion of the peptide is expected to bindto the MHC-II complex or the MHC-I complex in case ofcross-presentation.

Therefore, in a preferred embodiment, chemical addition of αGal epitopesis performed on amino acid residues corresponding to a “tag” regionadjacent to the amino acid sequence derived from the TAA.

In another embodiment, chemical addition of the αGal epitope isperformed to the N-terminal and/or C-terminal amino acid of theimmunizing peptide.

For the in vivo formation of immunocomplexes between anti-αGalantibodies and αGal⁽⁺⁾ TAA capable of complement activation, each Clmolecule must bind to at least two Fc sites for a stable CI-antibodyinteraction. Circulating IgM exists in a planar configuration and doesnot expose the Clq binding sites. IgM exposes its Clq binding sitesafter binding to an antigen on a membrane. For this reasonimmunocomplexes formed by anti-αGal IgM and soluble αGal⁽⁺⁾ TAA will notlikely activate the complement cascade. On the contrary, an IgG moleculecontains only a single Clq binding site in the CH2 domain of the Fcportion of the immunoglobulin, so that stable Clq binding is achievedonly when two IgG molecules are within 30-40 nm of each other in acomplex, thereby providing two Clq binding sites. In order to formparticulate immunocomplexes containing more than one anti-αGal IgG andone αGal⁽⁺⁾ TAA molecule, each TAA molecule has to contain more than asingle αGal epitope. This is easily achievable for proteins that havebeen chemically modified with αGal epitopes at their lysine and/orcysteine residues. However, for the particular case of αGal⁽⁺⁾ TAApeptides, it is important to provide amino acids that serve as anchoringpoints for the chemical addition of αGal epitopes and that do not formpart of the immunogenic portion of the peptide. Therefore, in anotherembodiment, αGal epitopes are chemically added in vitro to syntheticpeptides with a structure comprising: 1) a sequence of 1-20 amino acidsat its amino terminus that contains the acceptor amino acids for theαGal epitopes, 2) a central 7-20 amino acid sequence of a TAA epitopeknown to elicit an immunogenic CD4+ or CD8+T cell response, and 3) asequence of 1-20 amino acids at the C-terminus that contains acceptoramino acids for addition of a second αGral epitope. Thus, while it isnot intended that the invention be limited by the length of theαGal-modified TAA peptide, it is preferred that peptides that flank theTAA peptide of the present invention are less than twenty amino acids inlength_Preferably, the flanking peptides comprises 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acids, at least one of whichcomprises an αGal acceptor amino acid for addition of an ocGal epitope.In this synthesis method the epitope is directly linked to the aminoacid with no other glycosidic residues between the two, and the linkagewill depend on the type of cross-linker used sucks as maleimide wherethe epitope is added to cysteines, or succinimide where it will be boundto lysine and the primary N-terminal amino group, or glutaraldehydewhere it will bind to serine or threonine. It is postulated that thesedifferent epitope linkages will cause difference in binding capacity ofanti-αGal antibodies and their capacity to form immunocomplexes. Someantibodies will bind preferentially to chemical epitope and some willbind preferentially to epitopes added naturally by αGT.

Pharmaceutical Preparations

According to the invention, purified TAA proteins, protein fragments orpeptides modified to express αGal epitopes are used as eitherprophylactic or therapeutic vaccines to treat tumors. Thus the inventionalso includes pharmaceutical preparations for humans and animalscomprising αGal⁽⁺⁾ TAA proteins or peptides. Those skilled in themedical arts will readily appreciate that the doses and schedules ofpharmaceutical composition will vary depending on the age, health, sex,size and weight of the human and animal. These parameters can bedetermined for each system by well-established procedures and analysise.g., in phase I, II and III clinical trials and by review of theexamples provided herein.

For administration, the αGal⁽⁺⁾ TAA proteins can be combined with apharmaceutically acceptable carrier such as a suitable liquid vehicle orexcipient and an optional auxiliary additive or additives. The liquidvehicles and excipients are conventional and are commercially available.Illustrative thereof are distilled water, physiological saline, aqueoussolutions of dextrose and the like.

Suitable formulations for parenteral, subcutaneous, intradermal,intramuscular, oral or intraperitoneal administration include aqueoussolutions of active compounds in water-soluble or water-dispersibleform. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilicsolvents or vehicles include fatty oils for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, include for example, sodium carboxymethylcellulose, sorbitol and/or dextran, optionally the suspension may alsocontain stabilizers. Also, αGal⁽⁺⁾ TAA proteins or peptides can be mixedwith immune adjuvants well known in the art such as Freund's completeadjuvant, inorganic salts such as zinc chloride, calcium phosphate,aluminum hydroxide, aluminum phosphate, saponins, polymers, lipids orlipid fractions(Lipid A, monophosphoryl lipid A), modifiedoligonucleotides, etc.

In addition to administration with conventional carriers, activeingredients may be administered by a variety of specialized deliverydrug techniques which are known to those of skill in the art.

The invention will now be described with respect to the followingexamples; however, the scope of the present invention is not intended tobe limited thereby. All citations to patents and journal articles arehereby expressly incorporated by reference.

EXAMPLES Example 1 Production of Retroviral Vector Expressing αGT,pLNCKG

A 1,077 base pair (bp) fragment of murine αGT gene was PCR amplified bya forward primer, 5′-ACAAAAGCTTGACATGGATGTCAAGGGAAAAGTAAT-3′, (SEQ IDNO: 1) which contains a Kozak sequence to enhance the translation ofαGT, and a reverse primer, 5′-AATTATCGATTCAGACATTATTTCTAAC-3′ (SEQ IDNO: 2), and then cloned into the ClaI and HindIII sites of pLNCX toproduce pLNCKG retroviral′ vector (FIG. 2). This vector was transfectedinto the packaging cell line 293.AMIZ to generate the vector producercell line 293.AMIZ/LNCKG (Young and Link 2000). Transfected cells wereselected in presence of G418 and ZEOCIN™ antibiotic for two weeks. Mixedpopulation of selected cells was subcloned by limiting dilutions. Singlecell-derived VPC were screened for their ability to effectivelytransduce human epithelial cancer cell lines established from differenttissues. The clone which supernatant consistently yielded highesttransduction efficiency and αGT expression on a panel of humanepithelial cancer cell lines was identified and designated 293Z.CKG VPC.A master cell bank, working cell bank and production lot was generatedfor 293Z.CKG VPC was originated from one vial of the seed bank, expandedin flasks at 37° C.±1° C. in 5%±1% CO₂. The culture medium was RPMI-1640supplemented with 10% fetal bovine serum (FBS) and 2 mm L-glutamine.When the 293Z.CKG VPC reached sufficient density, the culture fluids(supernatant) are harvested, filtered, and pooled into a sterilecontainer. The pool is thoroughly mixed and then aseptically filled intolabeled, sterile plastic bottles. (Labels contain the product name, lotnumber and date of filling.) The fill bottles are frozen and stored ator below −60° C. Retrovirus-containing supernatants from 293Z.CKG VPCcan be used to transduce αGal” cell lines of human, monkey, mouse orhamster (CHO) origin, in order to establish the ocGal(+) cells used forexpression of recombinant TAA.

Example 2 Production of Lentiviral Vector Expressing αGT, pHSPA

A PCR fragment containing the human phosphoglycerate kinase (PGK)promoter was amplified from pHPEA3 (Mautino and Morgan 2002) usingprimers 5′-CAGGAATTCACGGGGTTGGGG-3′ (SEQ ID NO: 3) and5′-TGACGTACGATTAGCTT GATCATCCCCCTG-3′ (SEQ ID NO: 4), digested withBspEI (completely filled in) and EcoRI and cloned in the EcoRI-EcoRVrestriction sites of pLITMUS29 (New England Biolabs) to yield plasmidpLPGK. A PCR fragment of murine αGT gene was PCR amplified by a forwardprimer, 5′-ACAAAAGCTTGACATGGATGTCAAGGGA AAAGTAAT-3′ (SEQ ID NO: 5),which contains a Kozak sequence to enhance the translation of αGT, and areverse primer, 5′-ATTGGTACCTCAGACA′TTATTTCTAAC-3′ (SEQ ID NO: 6) andcloned in the HindIII-KpnI sites of pLPGK. The PGK-αGT expressioncassette was excised from pLPGK with EcoRI-KpnI and cloned in the samerestriction sites of pHCPE (Mautino and Morgan 2002). Infectious vectorparticles were produced by transient cotransfection into 3×10⁶ 293Tcells with 15 μg of vector pHSPA, 10 μg of HIV-1 helper packaging vectorpCMVΔR82 and 5 μg of pLTR-G encoding VSV glycoprotein. Supernatant wascollected 60 h after transfection and filtered through 0.45 μm mesh.

Example 3 Transduction of CHO Cells with LNCKG Retroviral Vector

CHO cells are extensively used to produce recombinant proteins andantibodies secreted into the culture medium (Werner et al. 1998).Despite being derived from a Chinese hamster which has a functional geneencoding αGT, CHO cells are αGal⁽⁻⁾ (Sharma et al. 1996). To generateαGal⁽⁺⁾ CHO cells, 2×10⁶ cells are transduced with 2 mL of supernatantcontaining the LNCKG retrovirus with an infectious titer of 2×10⁶transducing units/mL. Cells are selected for resistance to Neomycin by atwo-week selection in medium supplemented with G418 1 mg/mL. After thisperiod of selection, cells are stained for expression of the αGalepitope with a chicken anti-αGal polyclonal antibody and sorted byfluorescence activated cell sorting.

Example 4 Transduction of B16 Melanoma Cells with pHSPA RetroviralVector

B16 melanoma cells express the melanoma TAA gp75 and gp100 and be usedas a source for purification of ° Gale° TAA (Naftzger et al. 1996).Despite being derived from wild type C57B16 mice which have a functionalcopy of vGT gene these cells are αGal⁽⁻⁾ (Rossi et al. 2005a). Togenerate αGal⁽⁺⁾ B16 cells, 2×10⁶ cells were transduced with 10 mL ofsupernatant containing the pHSPA lentivirus with an infectious titer of−1×10⁶ transducing units/mL. Transduction of cells was carried out at amultiplicity of infection of 5 transfection units per cell in thepresence of 10 μg,/mL polybrene. Cells were stained for expression ofthe αGal epitope with a chicken anti-αGal polyclonal antibody and sortedby fluorescence activated cell sorting [FIG. 1].

Example 5 Purification of αGal⁽⁺⁾ gp75 from αGal⁽⁺⁾ B16 Cells

The gp75 antigen can be purified from αGal B16 cells using a protocoldescribed previously (Naftzger et al. 1996; Vijayasaradhi and Houghton1991). Briefly, cells are grown in DMEM 5% serum and harvested with EDTA2 mM without trypsin. Cells are washed in PBS and 10¹⁰ cells (˜50 g cellpellet) are resuspended in 400 mL of homogenization buffer (20 mMTris/HCl pH 7.5, 5 mM MgCl2; and protease inhibitor cocktail). Next,cells are homogenized and pelleted at 1000 g for 5 min, and thesupernatant is recentrifuged at 10000 g for 20 min. The membrane pelletis resuspended in 40 mL 50 mM Tris/HCl, 3 M KCl and 5 mM EDTA,rehomogenized and pelleted at 100000 g for 90 min. The pellet isresuspended in 25 mL of 50 mM Tris/HCl and 0.5% Na-deoxycholate andcentrifuged at 100000 g for 30 min. The buffer in the supernatant isexchanged by ultrafiltration dialysis to 2 mM CHAPS, 150 mM NaCl and 20mM Tris/HCl and applied to a Mono Q columns equilibrated in the samebuffer. The column is washed with 15 volumes of the equilibration bufferand bound proteins are eluted with a linear gradient of NaCl of 10 mM-1M. Fractions are assayed by ELISA using the monoclonal Ab TA99. Positivefractions are pooled together, dialyzed against 10 mM Tris/HCl, 1 mMCaCl₂, 1 mM MnCl₂ and cocktail of protease inhibitors and applied to aConcanavalin A-Sepharose column (10 mL) and washed with 50 ml loadingbuffer. Bound proteins are eluted with 250 mM methylmannopyranoside andthe fractions are assayed for the presence of gp75 by ELISA. Positivefractions are dialyzed against 10 mM Tris, 2 mM CHAPS and a cocktail ofprotease inhibitors and loaded onto an AFFI-GEI,-Hz (BioRad)immunoaffinity column containing TA99 monoclonal antibody equilibratedin 10 mM Tris/HCl, 150 mM NaCl and 2 mM CHAPS. The cross-linking of TA99to the AFFI-GEL®-matrix is performed according to the manufacturer'sinstructions. The column is washed sequentially with: 1) 50 mM HEPES pH7.5, 150 mM NaCl; 2) 50 mM HEPES1 M NaCl; 3) 50 mM HEPES, 150 mM NaCl, 2mM CHAPS. Bound gp75 is eluted with a buffer of 150 mM Glycine, pH 3.5and 2 mM CHAPS.

A similar process is employed to purify gp75 protein from αGal⁽⁻⁾ B16cells, to be used as negative controls in vaccination experiments.

Example 6

ELISA of αGal⁽⁺⁾ gp75

The assay to test for the presence of gp75 in column fractions isperformed by ELISA. Briefly, 200 μL of each sample is coated for 2 h onan ELISA plate. Wells are washed 6 times with TBS, and the wells areblocked for 1 h at room temperature with 300 μL 1,5% BSA in 150 mM NaCl.Plates are washed 6 times with 1×TBS, and TA99 mAb 100 μL (1:1000dilution) is added to each well and incubated for 30 min. After washing,a secondary biolinylated rabbit anti-mouse IgG2A is added (100 uL,1:1000 dilution) and incubated for 30 min. After 6× washing, 100 uL ofstreptavidin-horseradish (1:5000 dilution) peroxidase is added to eachwell and incubated for 30 min. After 6× washing, TMB-H₂O₂ is added toeach well and the reaction is stopped after 15-30 min with 4N H₂SO₄.

Example 7 Detection of αGal Epitopes on αGal⁽⁺⁾ gp75 by Western Blot

The presence of αGal epitopes on the affinity purified gp75 protein canbe tested by Western blot. Purified protein is run under denaturing andreducing conditions on SDS-PAGE, transferred to membranes and tested forbinding to either: 1) Bandeiraea simplicifolia IB4-HRP tectin(BS-lectin); 2) affinity-purified anti-αGal human antibodies; 3)anti-αGal chicken IgY, obtained by immunization of eggs with BSA-αGal (VLabs); or 4) the anti-αGal mouse monoclonal antibody M86 (IBM). Theanti-αGal human IgG polyclonal antibody is purified from normal humansera, by affinity chromatography on columns Sepharose columnscrosslinked to HSA-αGal (V Labs). Protein samples are separated on a3.6% polyacrylamide stacking gel and a 12% resolving gel for 40 min at200V. Proteins are then transferred to PVDF membranes by electroblottingand blocked overnight in 0.5% Tween-PBS (TPBS) with 1% bovine serum. Themembranes are then incubated in either human anti-αGal, anti-αGalchicken IgY (1:1000) or 20 ug ml/horseradish peroxidase (HRP)-conjugatedBS-lectin, both diluted in TPBS/1% bovine serum, for 1 h at roomtemperature followed by four washes for 15 min each in TPBS, The blotsare incubated with HRP-conjugated secondary antibodies either againsthuman IgG, chicken IgY or anti-mouse IgM antibody, diluted 1:1000, for30 min at room temperature and washed as above. Membranes are thenembedded in a solution of chemofluorescence substrate and then exposedto x-ray film.

Example 8 Expression of αGal⁽⁺⁾ Recombinant Fusion Proteins in αGal⁽⁻⁾Mammalian Cells

The following experiment was conducted to demonstrate that addition ofsecretory or Golgi amino acid sequence localization signals to a TAAthat does not normally traffick through the Golgi would result intrafficking and expression of such TAA in the right subcellularcompartment and with the proper glycosylation pattern (FIG. 2). Fourretroviral vectors based on Moloney marine: leukemia virus wereconstructed with expression cassettes encoding the following fusionproteins:

-   -   1] GFP-αGT    -   2] SLS-GFP-αGT    -   3] GLS-GFP-αGT    -   4] NLS-GFP-αGT        where SLS is the secretory localization signal from placental        alkaline phosphatase (amino acid sequence MDLLLLLLLGLRLQLSLGRIP        SEQ ID NO: 12), GLS is the Golgi localization sequence of ctGT        (MDVKGKVILLMLIVSTVVVVFWEYVNRIP SEQ ID NO: 13) and NLS is a        control nuclear localization signal (amino acid sequence        MDPKKKRKVRIADPKKKRKV SEQ ID NO:14). In these constructs, the        endogenous Golgi localization signal has been deleted from αGT.        Also, GFP is used instead of a true TAA to monitor the        localization of GFP protein by fluorescence microscopy. In this        particular case, the antigen is directly fused to αGT to ensure        co-expression of αGT and that of the antigen. These constructs        were produced in A375.AMIZ retroviral packaging cell line, and        the supernatant was used to transduce human A375 melanoma cells.        Activity of αGT was monitored by staining cells with IB4 lectin        from G. simplicifolia conjugated to fluorescein and analyzed by        FACS. The results indicate that if no subcellular localization        signal is added to the fusion protein, GFP and αGT are        coexpressed in the cytoplasm and there is no αGT enzymatic        activity as cells are αGal⁽⁻⁾ by FAGS. Addition of the SLS or        the GLS to the N-terminus of GFP results in the proper ER/Golgi        localization of the fusion protein, and αGT activity can be        measured as the transduced A375 cells are aGal⁽⁺⁾ by FACS.        Addition of a control NLS results in nuclear distribution of the        fusion protein and undetectable αGT activity as cells are        αGal⁽⁻⁾ by FACS. This example demonstrates that the proper SLS        or GLS has to be added to the N-terminus of the putative TAA to        be expressed in these cells in order to achieve proper cellular        trafficking and expression of αGT to mediate the addition of        αGal epitopes to the TAA of interest. Replacement of GFP by the        TAA sequence of interest in Construct #2 would allow one to        purify the aGal⁽⁺⁾ TAA from the culture supernatant. Addition of        a sequence affinity tag and protease cleavage site between the        TAA and αGT would allow to recover the aGal⁽⁺⁾ TAA free form the        αGT portion.

Example 9 Expression, Purification and Synthesis of etGal⁽⁺⁾ BORISProtein Fragment Vaccine

Among the different classes of TAA, cancer testis genes are excellentvaccine candidates as they are not normally expressed in anynon-malignant tissue of adult females and are only expressed in thetestis of adult males. As testes are an immunoprivileged tissue, cancertestis TAA is expected to be similar to non-self antigens and trigger apotent immune response. More than 80 cancer testis genes have beenidentified so far (Simpson et al. 2005). Cancer testis genes such asNY-ESO1, MAGE-A1 and BORIS are expressed in many cancers cell lines andprimary tumors. BORIS encodes a DNA binding protein that shares acentral 11-zinc finger domain with the epigenetic regulator ofimprinting CTCF (Loukinov et al. 2002). CTCF has a ubiquitous expressionprofile which does not make it a good candidate for vaccination. CTCFand BORIS differ in their N-terminus and C-terminus amino acid sequence.Moreover, CTCF and BORIS have been shown to reciprocally bind to thepromoter of cancer testis genes such as NY-ESO1 in pulmonary carcinomasand expression of BORIS is correlated with the expression of NY-ESO1(Hong et al. 2005). BORIS expression has been documented in cell linesand primary tumors of diverse histology such as prostate, lung, colon,breast, ovary, stomach, liver, glia, colon and esophagus (Lobanenkov etal. 2005). This makes the N-terminus and C-terminus domain of BORISmodified by addition of αGal epitopes and excellent candidate forantitumor vaccination for diverse types of cancers. In fact, DNAvaccination with a plasmid encoding BORIS lacking the DNA bindingdomain, in the presence of adjuvant and boosted with adenoviral vectorsencoding the same protein domains shows some antitumor effect in Balb/Cmice inoculated with 4T1 breast tumor cells, which express BORIS mRNA(Loukinov et al. 2006). However, none of the animals survived more than60 days which indicates that the vaccination protocol still needs to befurther optimized. Modification of the BORISA(ZF) by in vitro additionof αGal epitopes is expected to yield a highly immunogenic vaccine thattriggers both humoral and cellular antitumor immune response.

The following procedure describes the cloning of the N-terminal portionof human and murine BORIS genes into bacterial expression vectors. Onlythe portion encoding the N-terminal fragment of BORIS genes that do notencompass the 11-zinc finger domains that show high homology with CTCFis cloned into pTWIN1 expression vector (New England Biolabs).

The human N-terminal fragment of BORIS is obtained by RT-PCR from humanA375 cells using the primers hBORIS-F1 5′-GGTGGTCCATGGGTCGGGCAATGGCAGCCACTGAGATCCTCTGTCC-3′ (SEQ ID NO:7) and hBORIS-R1 5′-GGTGGTGGATCCTTAgtggtgGTGGTGGTGGTGGAAGG TTCCTTTTGCTCCCTT T-3′ (SEQ ID NO:8). Theseprimers amplify amino acids 1-258 of the human BORIS protein, whileadding a C-terminal (His)₆ tag to aid protein purification. The PCRfragment is digested with NcoI and BamHI and ligated into pTWINI vector(New England Biolabs) digested with NcoI and BamHI to yield plasmidpThBORIS-Nt.

The murine N-terminal fragment of BORIS is obtained by RT-PCR from 4T1cells using primers mBORIS-F1 5′-GGTGGTCCATGGGTCGGGCAATGGCTGCCGCTGAGGTCCCTGTCCCTT-3′ (SEQ ID NO: 9) and mBORIS-R1 5′-CTTAGTGGTGGTGGTGGTGGTGCTGAAAGCTCTGAGGCTTTCCCAA-3′ (SEQ ID NO: 10). These primersamplify amino acids 1-258 of the murine BORIS protein, while adding aC-terminal (His)₆ tag to aid protein purification. The PCR fragment isdigested with NcoI and ligated into pTWENI vector digested with NcoI andBamHI (completely filled-in with T4 DNA Polymerase), to yield plasmidpTmBORIS-Nt. Cloning of these fragments in the pTWIN1 vector creates anexpression cassette which consists in IPTG-inducible T7 RNA polymerasedependent promoter which drives the expression of RNA encoding a fusionprotein consisting of three domains: 1) a chitin binding domain, 2) aself cleaving Ssp Intein and 3) the BORIS₁₋₂₅₈₋His₆ domains. The vectorspThBORIS-Nt and pTmBORIS-Nt are transformed into E. coli BL21 pLysS. Anovernight culture in LB-Glucose is diluted 1:10 and grown at 37 C for4-6 h until OD₆₀₀ is ˜0.6. Protein expression is induced with IPTG 250μM for 1-3 h. Cells are washed in PBS and lysed by sonication in 20 mMPhosphate buffer pH 7, 200 mM NaCl, 1 mM EDTA, 0.1% TritonX 100 and 1 mMPMSF. Cell lysate is centrifuged at 18000 g for 30 min and thesupernatant is cleared by filtration through 0.45 μM pore. The clearedlysate is loaded onto a chitin column activated according to themanufacturer's instructions. The column is washed with 10 volumes of 20mM phosphate buffer pH 8.5, 600 mM NaCl, 1 mM EDTA, 0.1% Triton X100 and1 mM PMSF. Cleavage of the BORIS from the fusion protein is induced bychange in pH. Cleavage buffer (20 mM Phosphate buffer pH 6.0, 600 mMNaCl, 1 mM EDTA) is added to the column and incubated 16 h at roomtemperature. The cleaved BORIS protein is eluted by running 3 volumes ofcleavage buffer through the column. Protein is concentrated and dialyzedinto 50 mM Phosphate buffer pH 7 and loaded into a Ni⁺²-resin columnfollowing the manufacturer's instructions. The column is washed with 50mM imidazole and eluted in a linear gradient of 100-500 mM imidazole.Protein is concentrated and dialyzed by ultrafiltration into 50 mMPhosphate buffer pH 7.0. Yield and purity of the BORIS protein isperformed by ELISA and Western blot using an anti-His₆ monoclonalantibody following standard procedures.

To obtain the αGal⁽⁺⁾ N-terminal human and murine BORIS protein, a αGalepitope can be obtained from a commercial source (Dextra Labs, Reading,UK), or synthesized according to described Examples 22 to 32 orpublished protocols (Hanessian, 2001, Tetrahedron). This αGal epitope iscrosslinked to the purified protein by adding the αGal-NHS compounddissolved in DiVfF or DMSO at 10-20 fold molar excess, incubating for 2h at room temperature and dialyzing the αGal⁽⁺⁾ protein in PBS.

Example 10 Synthesis of αGal⁽⁺⁾ BORIS-Derived Peptides

An alternative embodiment for the generation of αGal⁽⁺⁾ BORIS vaccinesis to synthesize αGal⁽⁺⁾ peptides derived from the NH₂— or COOH—terminus of murine or human BORIS, excluding the amino acid sequencesthat are common to CTCF, followed by chemical addition of αGal epitopes.αGal epitopes can be obtained from a commercial source (Dextra Labs,Reading, UK), or synthesized according to described protocols (Hanessianet al. 2001b) or as described in Examples 22 to 32 and FIGS. 3-6. TheαGal-NHS epitope is crosslinked to primary amines in the peptides suchas Lysines or the free terminal NH₂— group. The αGal-Mal epitope iscrosslinked to free SH₂— groups such as the ones present in Cysteine orHomocysteine. Briefly, peptides are solubilized in DMSO at 10 mM anddiluted to 100 μM in 50 mM phosphate buffer pH 7. The αGal-NHS orαGal-Mal compounds are dissolved in DMF or DMSO at 10 mM. 50-100 μL ofαGal-NHS or αGal-Mal is added to the peptide solution (10-20 fold molarexcess) and the reaction is incubated at room temperature for 2 hours.Excess of unreacted αGal-NHS or αGal-Mal is eliminated by dialysis orultrafiltration.

The peptides used for vaccination have the structureαGal-Z-X₁₋₂₀-Peptide-X₁₋₂₀-Z-αGal, where Z can be a Cysteine,Homocysteine or Lysine, X₁₋₂₀ refers to any sequence of 1-20 aminoacids, and Peptide refers to the sequences of amino acids for the humanor murine BORIS indicated in Table 1. This table suggests differentpeptides that will be better suited to bind to different alleles ofMI-IC or HLA molecules:

TABLE 1 BORIS derived peptides to elicit antitumor response HumanHLA Type Peptide SEQ ID NO HLA-A201 VLSEQFTKI 20 VLTVSNSN 21 ILTLQTVHFT22 SVLEEEVELV 23 SVLEEEVEL 24 KLAVSLAET 25 LLAERTKEQL 26 HLA-A1LAETAGLTK 27 FIL A-A03 SVLSEQFTK 28 SLAETAGLIK 29 ILKEATKGQK 30EAANGDEAA A 31 LKEATKGQK 32 VLAPSEESEK 33 HLA-A24 LYSPQEMEVL 34 HLA-A26EQFTKIKEL 35 EVDEGVTCE 36 EESEKYILTL 37 HLA-A68.1 GVCREKDHR 38 SVLSEQFTK39 NVMVASEDSK 40 FVETMSGDER 41 HLA-B08 AERTKEQL 42 TRKR KQTI 43 HLA-B18QEMEVLQF 44 HLA-B2705 ERTKEQLFF 45 HLA-B4402 EESEKYILTL 46 HLA-DRB1VQVVVQQPGPGLLWL 47 LLSIQQQEGVQVVVQ 48 LLWLEEGPRQSLQQC 49 VETMSGDERSDEIVL50 GEMFPVACRETTARV 51 SEQFTKIKELELMPE 52 KLAVSLAETAGLIKL 53EMEVLQFHALEENVM 54 Murine MHC Peptide SEQ ID NO H2-K^(d) LYPPEELQRI 55SFQDPEHETL 56 HFHLLRENVL 57 YFTQIKEQICL 58 LWLDPEPQL 59 HFHLLRENV 60APVESDRRI 61 LQLPSVLWL 62 VTVSIPEEL 63

Example 11 Synthesis of αGal⁽⁺⁾ bcr-abl Derived Peptides for VaccinationAgainst Chronic Myelogenous Leukemia

Chronic myelogenous leukemia is generated by a translocation t(9; 22)that results in the chimeric bcr-abl gene encoding a 210 kDa fusionprotein. There are two possible fusions that result in a functionalin-frame Bcr-Abl fusion protein, characterized by the fusion of thesecond or third exon of Bcr and the second exon of Abl. These twofusions are designated b3a2 and b2a2. The junction of these two proteindomains generates a new amino acid sequence against which the patient isnot supposed to have been immunologically tolerized. Therefore, thisjunction sequence constitutes a novel TAA and a good candidate forvaccination approaches to eliminate residual disease in patients thatrespond to Imatinib or to treat patients that are resistant to Imatinib(Gleevec). Several studies have identified different peptides derivedfrom the Bcr-Abl fusion that bind to different alleles of class I and IIHLAs (Bocchia et al. 1996; Bocchia et al. 1995). Clinical trials havebeen conducted by vaccination with synthetic peptide vaccines (Cathcartet al. 2004; Pinilla-Ibarz et al. 2000). bcr-abl-derived peptide vaccinecan be safely administered to patients with CML and can elicit areliable specific CD4 immune response. However, no cytotoxic Tlymphocytes have been identified in these trials. A way to circumventthe poor immunogenicity of these peptides in order to elicit a morepotent CD8⁺ immune response would be by the methods of the presentinvention, consisting in the chemical modification of such peptides withαGal epitopes. The peptides described in the following Table could beused for vaccination against CML:

HLAType Peptide (SEQ ID NO:) HISA-A201 SSKALQRPVC-αGal 64αGal-CSSKALQRPVGSSICALQRPVGSSICALQRPVC-αGal 65 HLA-A3 KQSSKALQRC-αGal 66αGal-CKQSSICALQRGSICQSSICALQRGSICQSSICALQRC-αGal 67 HLA-A11ATGFKOSSICC-αGal 68 αGal-A TGFKQSSKCGSATGFKQSSKCGSATGFICQSSI(C-αGal 69HLA-A3/11 HSATGFKQSSIC-αGal 70αGal-CHSATGFICQSSICGSHSATGFKQSSKGSHSATGFKQSSKC-αGal 71 HLA-B8GFKQSSICALC-αGal 72 αGal-CGFICQSSKALCGSCGFICQSSKALCGSCGRKQSSICALC-αGal73 Class II αGal-CIVHSATGFKQSSICALQRPVASDFEPC-αGal 74

Example 12 Synthesis of αGal⁽⁺⁾ gp75-Derived Peptides

The following peptides, chemically synthesized by solid phase synthesis,can be used to test the immunogenicity of αGal⁽⁺⁾ peptides derived fromthe mouse TAA gp75 (TRP-1) in an αGT knockout mouse.

-   -   1] TWHRYHLL (SEQ ID NO: 75) is the natural sequence        corresponding to amino acids 222-229 of marine gp75    -   2] TAYRYHLL (SEQ ID NO: 76) is a heteroclitic variant of peptide        #1 that has better binding affinity to MHC-I H2-K^(b) (Dyall et        al. 1998)    -   3] KTAYRYEILL (SEQ ID NO: 77) is peptide #2 with an N-terminal        Lysine    -   4] KTAYRYHLLGSTAYRYHLL (SEQ ID NO:80) is derived from peptide #2        with an N-terminal tag containing an N-terminal Lysine    -   5] KTAYRYHLLGSTAYRYHLLGSTAYRYHLLK (SEQ ID NO: 78) is derived        from peptide #2 with an N-ter and C-ter tag consisting of the        sequence of peptide #2 flanked by N-ter and C-ter Lysines that        serve as anchoring points for chemically synthesized αGal        epitopes with NHS linkers.

If the N-ter of each peptide is not blocked by N-formylation, then theN-terminal Lys can be omitted from the sequence of the peptides.

Chemically activated αGal epitopes (of generic formula αGal-L1-NHS,wherein LI is a linker) can be obtained from a commercial source (DextraLabs, Reading, UK), or synthesized according to described protocols(Hanessian et al. 2001b) or as described in Examples 22 to 32 and FIGS.3-6.

This αGal-L1-NHS epitope is crosslinked to peptides #3, #4 and #5 usingthe following method. Briefly, peptides are solubilized in DMSO at 10 mMand diluted to 100 11M in 50 mM phosphate buffer pH 7. The αGal-NHScompound is dissolved in DMF or DMSO at 10 mM. 50-100 μL of αGal-NHS isadded to the peptide solution (10-20 fold molar excess) and the reactionis incubated at room temperature for 2 hours. Excess of unreactedαGal-NHS is eliminated by dialysis.

Example 13 Induction of Anti-αGal Antibodies in αGT KO Mice byImmunization with Rabbit Red Blood Cells

Females and males 8 to 14 weeks old αGT knockout mice were used in thisstudy. Mice were initially of mixed haplotype (H-2 b/d) and by breedingand selection the current colony of αGT KO mice was obtained consistingin F4 inbreeding generation of H-2 b/b haplotype. These animals producelow titers of natural antibodies against αGal epitopes. To increase thetiter of anti-αGal Ab mice are immunized intraperitoneally (i.p.) with1×10⁸ rabbit red blood cells twice, two weeks apart. The titers ofanti-αGal Ab are checked one week after the last RRBC injection tocorroborate that all mice in the study have high titers of anti-αGal Ab.In this manner, this model mimics the high titer of natural anti-αGalantibodies present in humans. All mice used in this study have highanti-αGal

Ab titers greater than 1:500 dilution, measured by ELISA.

Example 14 Immunization with αGal⁽⁺⁾ gp75 Purified from αGal⁽⁺⁾ B16Cells

The following animal experiment is performed to induce antitumorimmunity against B16 melanoma cells with αGal⁽⁺⁾ gp75 protein purifiedfrom αGal⁽⁺⁾ B16 melanoma cells. αGT KO mice (of C57B1/6 geneticbackground, H-2K^(b/b)) are immunized with rabbit red blood cells (RRBC)as described previously to induce the presence of anti-αGal antibodies.Additionally, wild type C57B16 mice, which do not develop anti-αGalantibodies, are used as control groups. Each animal is immunized with 2to 3 doses of 1 to 100 μg of purified αGal⁽⁺⁾ or αGal⁽⁺⁾ gp75,resuspended in saline solution, without adjuvant. Examples of possibletreatment and control groups and doses are:

Group Strain Vaccine Dose 1 αGT KO saline — 2 αGT KO αGal⁽⁻⁾ gp75  1 ug3 αGT KO αGal⁽⁻⁾ gp75  10 ug 4 αGT KO αGal⁽⁻⁾ gp75 100 ug 5 αGT KOαGal⁽⁺⁾ gp75  1 ug 6 αGT KO αxGal⁽⁺⁾ gp75  10 ug 7 αGT KO αGal⁽⁺⁾ gp75100 ug 8 C57B16 αGal⁽⁺⁾ gp75 100 ug 9 C57B16 αGal⁽⁻⁾ gp75 100 ug

The vaccines are administered by subcutaneous injection, and each doseis administered 7-10 days apart. Immunologic tests are conducted oneweek after the last immunization as described below.

Example 15 Immunization with αGal⁽⁺⁾ gp75-Derived Peptides

The following immunizations are performed to induce antitumor immunityagainst B16 melanoma cells with αGal⁽⁺⁾ gp75-derived peptides. αGT KOmice (of C5781/6 genetic background, H-2K^(b/b)) are immunized withrabbit red blood cells (RRBC) as described previously to induce thepresence of natural anti-αGal antibodies. Additionally, wild type C57B16mice, which do not develop anti-αGal antibodies are used as controlgroups. Each animal is immunized with 2 to 3 doses of 1 to 10 μg ofpurified αGal⁽⁺⁾ or αGal⁽⁻⁾ gp75-derived peptide, resuspended in salinesolution, with or without adjuvant. Examples of possible treatment andcontrol groups and doses are:

G# Strain Peptide Vaccine(SEQ ID NO:) Dose  1 αGT KO none —  2 αGT KOαGal⁽⁻⁾-KTAYRYHLL(79)  1 ug  3 αGT KO αGal⁽⁻⁾-KTAYRYHLL(79) 10 ug  4αGT KO αGal⁽⁺⁾-KTAYRYHLL(79)  1 ug  5 αcGT KO αGal⁽⁺⁾-KTAYRYHLL(79)10 ug  6 αGT KO αGal⁽⁻⁾-KTAYRYHLLGSTAYRYHLL(80)  1 ug  7 αGT KOαGal⁽⁻⁾-KTAYRYHLLGSTAYRYHLL(80) 10 ug  8 αGT KOαGal⁽⁺⁾-KTAYRYHLLGSTAYRYHLL(80)  1 ug  9 αGT KOαGal⁽⁺⁾-KTAYRYHLLGSTAYRYHLL(80) 10 ug 10 αGT KOαGal⁽⁻⁾-KTAYRYHLLGSTAYRYHLLGSTAYRYHLLK(81)  1 ug 11 αGT KOαGal⁽⁻⁾-KTAYRYHLLGSTAYRYHLLGSTAYRYHLLK(81) 10 ug 12 αGT KOαGal⁽⁺⁾-KTAYRYHLLGSTAYRYHLLGSTAYRYHLLK(81)  1 ug 13 αGT KOαGal⁽⁺⁾-KTAYRYHLLGSTAYRYHLLGSTAYRYHLLK(81) 10 ug 14 C57B16αGal⁽⁻⁾-KTAYRYHLL(79) 10 ug 15 C57B16 αGal⁽⁺⁾-KTAYRYHLL(79) 10 ug 16C57B16 αGal⁽⁻⁾-KTAYRYHLLGSTAYRYHLL(80) 10 ug 17 C57B16αGal⁽⁺⁾-KTAYRYHLLGSTAYRYHLL(80) 10 ug 18 C57B16αGal⁽⁻⁾-KTAYRYHLLGSTAYRYHLLGSTAYRYHLLK(81) 10 ug 19 C57B16αGal⁽⁺⁾-KTAYRYHLLGSTAYRYHLLGSTAYRYHLLK(81) 10 ug

The vaccines are administered by subcutaneous or intradermal injection,and each dose is administered 7-10 days apart. Immunologic tests areconducted one week after the last immunization as described below.

It has been previously described that in the presence of adjuvant, theheteroclitic peptide TAYRYHLL (SEQ ID NO:76) induces protective immunityagainst B16 melanoma in C57B1/6 mice (H-2K^(b)), as this tumor cell lineexpresses gp75 and the peptide TWHRYHLL (SEQ ID NO:75) in the context oftheir MHC-I molecules (Dyall et al. 1998). However, in the absence ofadjuvant this peptide is not immunogenic. The presence of αGal epitopeselicits the formation of immunocomplexes, which are able to elicit animmune response even in the absence of adjuvant. Analysis of the immuneresponse parameters obtained after the immunization treatments describedabove will give information regarding the effect of the αGal epitope onthe immunogenicity of the peptide, the effects of the αGal epitope onthe potency or dose necessary to achieve certain levels of immuneresponse and the effect of the presence of anti-αGal antibodies on thefinal immune response. Additionally, it will evaluate the effects ofhaving none, one or two αGal epitopes per molecule and the effects ofhaving the αGal epitope immediately linked to the immunizing peptide orseparated from the αGal epitope by a tag of 8-11 amino acids.

Example 16 Evaluation of Immune Response in Mice after Vaccination withαGal⁽⁺⁾ gp75 or αGal⁽⁺⁾ gp75-Derived Peptides

It is expected that after immunization with gp75 protein or peptides Tcells will show a higher response in the ability to recognize αGal⁽⁻⁾B16 cells that express gp75 when the immunizing antigen is αGal⁽⁺⁾ thanwhen the immunizing antigen is αGal⁽⁻⁾. To test this hypothesis,splenocytes from mice vaccinated with αGal⁽⁺⁾ or αGal⁽⁻⁾ gp75 or peptidevaccines are harvested and cultured for 6 h in presence or absence ofstimulation. The control for maximum stimulation is the ionophorePMA/Ca⁺⁺. 10⁶ splenocytes are cultured with 10⁵ irradiated B16 cells tomeasure specific recognition or with CA320M intestinal sarcoma, anon-specific αGal⁽⁺⁾ cell line with identical H-2^(b/b) haplotype. Thiscell line was obtained by intraperitoneal injection of 2 mg9,10-dimethyl-1,2-benz-anthracene (DMBA) and 1 mg 3-methylcholanthrene(3-MC) dissolved in 250 μl of olive oil at two week intervals into αGTKO mice. Alternatively, as control for specificity of immunity mediatedagainst this peptide, the B16-derived radiation induced gp75⁽⁻⁾ cellB78H.1 can be used as a negative control (Mintz and Silvers 1967). Afterincubation cells are harvested and stained for intracellular IFNγ and/orTNFα. Detection is performed by FACS gating for lymphocytes in theforward scatter plot. The percentage of lymphocytes activated byPMA/Ca++ ionophore is considered the maximum activation detectable inthis experiment. Resting (unstimulated) T cells and T cells stimulatedwith CA320M or B78H.1 are expected to have undetectable intracellularIFNγ or TNF-α, indicating that no T cells precursors that are able torecognize antigens in CA320M or other antigens on B16 that are notderived from gp75 are induced after vaccination. On the contrary,vaccination with αGal⁽⁺⁾ gp75 or αGal⁽⁺⁾ gp75-derived peptides isexpected to induce T cell precursors that specifically recognize αGal⁽⁻⁾B16 in vitro. Additionally, the number of precursors in spleens frommice vaccinated with αGal⁽⁺⁾TAA is expected to be superior than thenumber of precursors observed in spleens of mice vaccinated with αGal⁽⁻⁾TAA. This result would suggest that these T cells induced aftervaccination with αGal⁽⁺⁾ TAA maybe responsible for tumor prevention inmice challenged with B16 tumor cells.

In a different set of experiments, cell-surface activation markers canbe used to measure specific T cell recognition of the αGal⁽⁻⁾B16melanoma cell line induced by vaccination. It is well described thatupon engagement of the T cell receptor (TCR), T cells up-regulateseveral cell surface molecules that indicate an activated state of thelymphocyte. One of those molecules is the IL-2 receptor a chain or CD25.Upon TCR engagement, CD25 is up-regulated and can be detected by FACS at1 day after activation. Similarly, CD69 (or very early activationantigen (VEA)) is up-regulated upon T cell activation. CD69 functions asa signal-transmitting receptor in different cells, it is involved inearly events of lymphocyte activation and contributes to T cellactivation by inducing synthesis of different cytokines, and theirreceptors. Both activation markers (CD25 and CD69) are expressed at verylow level in resting T cells. To demonstrate that vaccination withαGal⁽⁺⁾ TAA proteins or peptides induced T cell precursors able torecognize specifically B16, the up-regulation of activation markers canbe used as parameters to measure recognition and activation. Cells areharvested from the spleens of mice vaccinated with αGal⁽⁻⁾ or αGal⁽⁺⁾gp75 or gp75-derived peptides. These cells are cultured withoutstimulation or stimulated with a negative control cell line (CA320M) orwith αGal⁽⁺⁾ B16. After 24 hours of culture, cell are harvested andstained to detect CD25 or CD69 by FACS. It is expected that resting Tcells (no stimulation) and cells stimulated with the syngeneicnon-melanoma cell line CA320M expressed very low levels of activationmarkers. On the other hand, increased numbers of activated (CD25⁽⁺⁾ andCD69⁽⁺⁾) lymphocytes from mice vaccinated with αGal⁽⁺⁾ TAA is expectedwhen T cells are cultured with αGal⁽⁺⁾ B16.

Example 17 Prevention of Subcutaneous Melanoma Tumor Growth byVaccination with αGal⁽⁺⁾ TAA or αGal⁽⁺⁾ TAA-Derived Peptides

In order to test whether vaccination with αGal⁽⁺⁾ TAA or TAA-derivedpeptides induces protective antitumor immunity, different αGal⁽⁺⁾ orαGal⁽⁻⁾ peptides derived from gp75 are used to vaccinate αGT KO micethat have been primed to have anti-αGal antibodies by vaccination withRRBC. Peptides #1 to #5 are synthesized in their αGal⁽⁺⁾ or αGal⁽⁻⁾forms and different groups of 15 αGT KO mice each receive three weeklydoses of 5 μg of a peptide vaccine, injected subcutaneously withoutadjuvant. One week after the last dose of vaccination mice arechallenged by subcutaneous (s.c.) injection of 10⁵ B16 cells. Tumorgrowth is monitored 3 times a week with a Vernier caliper by measuringthree perpendicular diameters, which are multiplied to obtain theminimum cube that will contain the tumor. When tumors reach a volumehigher than 1000 mm³, animals are sacrificed. Differences in the potencyof each vaccine is evaluated by statistical comparisons of the survivalcurves (Kaplan-Meier) by the Logrank test, and also by statisticalevaluation of differences in the parameters that describe tumor growthkinetics such as time of tumor onset (defined as the time it takes for atumor to achieve an irreversible volume higher than a predeterminedthreshold (such as 65 mm³) and tumor growth rate (assuming exponentialgrowth kinetics).

Example 18 Prevention of Metastatic Melanoma Tumor Growth by Vaccinationwith αGal⁽⁺⁾ TAA or αGal⁽⁺⁾ TAA-Derived Peptides

In order to test whether vaccination with αGal⁽⁺⁾ TAA or TAA-derivedpeptides induces protective antitumor immunity that will restrain thegrowth of metastatic tumor nodules, different aGal⁽⁺⁾ or αGal⁽⁻⁾peptides derived from gp75 are used to vaccinate αGT KO mice that havebeen primed to have anti-αGal antibodies by vaccination with RRBC.Peptides #1 to #5 were synthesized in their αGal⁽⁺⁾ or αGal⁽⁺⁾ forms anddifferent groups of 15 αGT KO mice each receive three weekly doses of 5pig of a peptide vaccine, injected subcutaneously without adjuvant. Oneweek after the last dose of vaccination mice are challenged by i.v.injection (in the tail vein) of 5×10⁴ B16 cells in 0.1 mL of salinesolution. Metastatic tumor burden is measured 3-4 weeks after tumorchallenge by sacrificing the animals and measuring lung weight, wholeweight of metastatic tumor nodules outside the lung (intestine, liver,lymph nodes), and by measuring the ratio of melanin/total protein inhomogenates of tissue containing tumor nodules. Melanin is measured byhomogenizing tissue in NaOH 1N (5 g of unfixed tissue per 5 mL of NaOH1N), in a grinder followed by sonication. The homogenate is centrifugedat 1000 g for 10 min and the supernatant is filtered through 0.45 μmfilters. Pure melanin (Sigma) is used to prepare a standard curve, andabsorbance of the filtered supernatant is determined at 405 nm. Totalprotein is also determined in the supernatant by a regular BCA assay.The ratio of melanin to total protein is a measure of the tumor burdenwithin the tissue bearing metastatic nodules.

Example 19 Treatment of Pre-Established Metastatic Melanoma Tumors byVaccination with αGal⁽⁺⁾ TAA or αGal⁽⁺⁾ TAA-Derived Peptides

In order to test whether vaccination with αGal⁽⁺⁾ TAA or TAA-derivedpeptides is potent and fast enough to induce antitumor immunity able totreat 4-5 day old pre-established B16 tumors, different αGal⁽⁺⁾ orαGal⁽⁻⁾ peptides derived from gp75 are used for vaccination of aGT KOmice that have been primed to have anti-αGal antibodies by vaccinationwith RRBC. One week after the last RRBC immunization, aGT KO mice areinjected subcutaneously with 10⁵ B16 live cells. Four to five days aftertumor challenge mice receive three weekly doses of vaccination witheither peptide #1 to #5 in their αGal⁽⁺⁾ or αGal⁽⁺⁾ forms. Tumor growthis monitored 3 times a week with a Vernier caliper by measuring threeperpendicular diameters, which are multiplied to obtain the minimum cubethat will contain the tumor. When tumors reach a volume higher than 1000mm³, animals are sacrificed. Differences in the potency of each vaccineis evaluated by statistical comparisons of the survival curves(Kaplan-Meier) by the Logrank test, and also by statistical evaluationof differences in the parameters that describe tumor growth kineticssuch as time of tumor onset (defined as the time it takes for a tumor toachieve an irreversible volume) and tumor growth rate (assumingexponential growth kinetics).

Example 20 Treatment of Pre-Established Melanoma Tumors by Adoptive TCell Transfer from Mice Vaccinated with αGal⁽⁺⁾ TAA or αGal⁽⁺⁾TAA-Derived Peptides

The in vitro experiments shown above indicate that more quantity andquality of melanoma specific T cells are induced in mice vaccinated withaGal⁽⁺⁾ TAA than in mice receiving αGal⁽⁻⁾ TAA vaccination. Thesemelanoma specific T cells are expected to be increased in numbers (moreT cells found in spleens) and to produce more TNFα. Also, moresplenocytes are expected to be activated when co-cultured with B16(up-regulation of CD25 and CD69). It is expected that mice bearing bothsubcutaneous and lung pulmonary metastases receiving αGal⁽⁺⁾ TAAvaccines show prolonged survival and increased clearance of the lungtumors than mice receiving αGal⁽⁻⁾ TAA vaccines. These two groups ofdata would indicate that T cells induced by αGal⁽⁺⁾ TAA vaccination areresponsible for the treatment of pre-established melanoma tumors.However, it is not obvious that this is the case since it has been shownthat large amount of melanoma-specific T cells are insufficient to treatpre-established subcutaneous melanoma tumors, since they are in atolerant state (Overwijk et al. 2003). We hypothesized that vaccinationwith αGal⁽⁺⁾TAA would induced a strong T cell mediated immunity that canbe rapidly activated upon recall to mediate tumor clearance in micebearing pre-established disease. To demonstrate this hypothesis adoptivecell transfer experiments have to be conducted. Donor mice arevaccinated with three doses of αGal⁽⁺⁾ or αGal⁽⁻⁾ TAA. Recipient miceare injected i.v with 10⁵ live αGal⁽⁺⁾ B16 to establish the lungmelanoma metastases and randomized. Four days after i.v injection ofnon-irradiated B16, mice receive, or not T cells from donors vaccinatedwith αGal⁽⁺⁾ or αGal⁽⁻⁾ TAAs. Four weeks later, the lung melanomametastasis burden is measured by enumerating lung tumors, by weightinglungs obtained in block and by quantification of melanin/protein ratiosin homogenates of lung tissue. Melanin is measured by A₄₀₅ nm. A similarexperiment was performed previously using irradiated otGal(4) or αGal⁽⁻⁾B16 whole cell vaccines and enhanced antitumor response was observed byvaccination with αGal⁽⁺⁾ whole cell vaccines (Rossi et al. 2005a).Similarly, the same outcome is expected by vaccination with αGal(4) TAAprotein or peptides.

Example 21 Antitumor Vaccination with αGal⁽⁺⁾ BORIS-Derived Peptides

In order to test the effect of immunization of mice with αGal⁽⁺⁾BORIS-derived peptides the following αGal⁽⁺⁾ peptide is synthesized:

(SEQ ID NO: 11) KLYPPEELQRIGSLYPPEELQRIGSLYPPEELQRIK

This peptide is modified by the chemical addition of any αGal epitopeGalα1-3Galβ31-4GlcNAc-R₁-NHS as described below in Examples 22 to 32.

αGT KO mice bred in the C57B1/6 genetic background are primed by 2 or 3intraperitoneal injections of 10⁸ RRBC to induce the production ofanti-αGal antibodies. On week after the last RRBC immunization, animalsreceive three weekly subcutaneous immunizations of 5 μg of αGal⁽⁺⁾ BORISpeptide without adjuvant. One week after immunization animals receive asubcutaneous injection of 10⁵ B16 cells in 100 μL saline and tumorgrowth is monitored over time. Animals are sacrificed when tumor volumeis higher than 1000 mm³.

Example 22 Synthesis of αGal EpitopeGalα1-3Galα1-4Glc-L₁-N-hydroxysuccinimide Ester (NLG-αGal-001)

All commercial reagents and solvents were used as received withoutfurther purification. The reactions were monitored using thin layerchromatography using 0.25 mm EM Science silica gel plates (60E-254). Thedeveloped TLC plates were visualized by immersion in potassiumpermanganate solution followed by heating on a hot plate. Flash columnchromatography was performed with Fisher Scientific silica gel grade 60,230-400 mesh. 1H NMR spectra were obtained with a Bruker DRX400 andVarian VXR300 respectively. 1H NMR spectra were reported in parts permillion (ppm) relative to CDCl3 (7.27 ppm) and CD3OD (4.80 ppm) as aninternal reference.

A bifunctional linker was designed such that there is a secondaryaminooxy group on one end and a carboxylic acid on the other endconformed by the structure COOH—R₁—O—NH—CH₃ wherein R₁ is any linear orbranched alkyl group of 1 to 30 carbon atoms, wherein one or more carbonatoms in such alkyl group can be substituted by O, S, or N and whereinone or more hydrogens can be substituted by hydroxyl, carbonyl, alkyl,sulphydryl or amino groups. As shown in FIG. 3, the amine linker 1.4 waschosen for initial studies. The linker was synthesized in 2 steps fromknown compound N-boc-N-methylhydroxylamine. The aminooxy linker istreated with α-Gal in DMF:AcOH (1:1) to produce glycosylated product1.5. Activation of carboxylic acid with NHS (N-hydroxysuccinimide)results in the formation of activated α-Gal epitope NLG-αGal-001.

Example 23 Synthesis of αGal epitopeGalα1-3Galα1-4Glc-L₁-N-hydroxysuceinimide ester (NLG αGal-002)

The αGal epitope NLG-αGal-002 was designed with an ester group withinthe linker region in order to facilitate the removal of this αGaltrisacharide by intracellular esterases, following the synthesis schemedescribed in FIG. 4. The bifunctional linker 2.3 was synthesized in 4steps from N-methylhydroxylamine. Glycosylation of α-Gal with theaminoxy linker 2.3 followed by activation of the terminal carboxylicacid with NHS would produce the desired α-Gal epitope.

Example 24 Synthesis of N-methyl-N-boc-(2-hydroxypropyl)hydroxylamine(Compound 2.1)

To a solution of N-methyl-N-Boc hydroxylamine (Beshara et. al. Org.Lett. 2005, 7, 5729 (444 mg, 3.02 mmol) and propylene oxide (0.25 mL,162 mmol) in EtOH (8 mL) at room temperature was added K2CO3 (458 mg,3.32 mmol). The reaction mixture was further stirred at room temperaturefor 16 h. The solvent was removed in vacuo and the crude product waspurified by silica gel flash column chromatography using 25%EtOAc/hexane as eluent to yield the corresponding secondary alcohol ascolorless oil (241 mg, 1.18 mmol, 39%). ¹H NMR (CDCl₃, 400 MHz): δ (ppm)1.08 (d, 3H, J=6.4 Hz), 1.45 (s, 9H), 3.05 (s, 3H), 3.45 (dd, 1H, J=9.6,11.6 Hz), 3.78 (dd, 1H, J=2.4, 11.2 Hz), 3.90-3.96 (m, 1H), 4.17 (br s,1H).

Example 25 Synthesis of2,2,5,8-tetramethyl-4,10-dioxo-3,6,9-trioxa-5-azamidecan-13-oic acid(Compound 2.2)

To a solution of N-methyl-N-boc-(2-hydroxypropyl)hydroxylamine (227 mg,1.11 mmol) in dichloromethane (4 mL) were added DMAP (41 mg, 0.333 mmol)and succinic anhydride (167 mg, 1.67 mmol). The resulting mixture wasstirred for 20 h at room temperature. The reaction mixture was pouredinto a saturated solution of ammonium chloride (15 mL) and extractedwith DCM (3×40 mL). The combined organic layer was washed with water anddried over sodium sulfate. The solvent was removed in vacuo and thecrude product was purified by silica flash column chromatography using40% EtOAc/hexane as eluent to give the product as a white solid (227 mg,0.744 mmol, 67%). ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 1.14 (d, 3H, J=6.4Hz), 1.45 (s, 9H), 2.51-2.57 (m, 4H), 2.91 (s, 3H), 3.77-3.79 (m, 2H),5.04-5.11 (m, 1H).

Example 26 Synthesis of4-(1-(methylaminooxy)propan-2-yloxy)-4-oxobutanoic acid (Compound 2.3)

2,2,5,8-Tetramethyl-4,10-dioxo-3,6,9-trioxa-5-azamidecan-13-oic acid(227 mg, 0.744 mmol) was dissolved in 4 mL TFA. The reaction mixture wasstirred at room temperature for 4 h and concentrated. The crude productwas purified by silica gel flash column chromatography using 12%MeOH/dichloromethane as eluent. The desired product was obtained as acolorless gel (30 mg, 0.146 mmol, 20%). ¹H NMR (CD₃OD, 300 MHz): δ (ppm)1.17 (d, 3H, J=6.6 Hz), 2.58 (s, 3H), 2.61 (s, 3H), 3.66 (d, 2H, J=5.4Hz), 5.10-5.16 (m, 1H).

Example 27 Synthesis of αGal epitopeGalα1-3Galα1-4Glc-L₁-N-hydroxysuccinimide ester (NLG αGal-003)

A different linker bearing an ester functionality in a differentposition of the linker can be formed by reacting hydroxylamine directlywith succinic anhydride. As described in FIG. 5, treatment ofN-methyl-N-boc hydroxylamine with succinic anhydride and DMAP resultedin carboxylic acid 3.1. Deprotection of Boc gave amine 3.2 in 100%yield. Conjugation of the linker with the αGal trisacharide wasperformed as described above for the synthesis of NLG-αGal-001 andNLG-αGal-002.

Example 28 Synthesis of4-(tert-butoxycarbonyl(methyl)aminooxy)-4-oxobutanoic acid (Compound3.1)

To a solution of N-methyl-N-boc hydroxylamine3 (536 mg, 3.65 mmol) inichloromethane (8 mL) were added DMAP (134 mg., 1.10 mmol) and succinicanhydride (548 mg, 5.48 mmol). The resulting mixture was stirred for 16h at room temperature. The reaction mixture was poured into a saturatedsolution of ammonium chloride (10 mL) and extracted with ethyl acetate(2×40 mL). The combined organic layer was washed with water (20 mL) anddried over sodium sulfate. The solvent was removed in vacuo and thecrude product was purified by silica flash column chromatography using45% EtOAc/hexane as eluent to give the corresponding acid as a whitesolid (693 mg, 2.81 mmol, 77%). ¹H NMR (CDCl₃, 400 MHz): 8 (ppm) 1.42(s, 9H), 2.65-2.70 (m, 4H), 3.17 (s, 3H).

Example 29 Synthesis of 4-(methylaminooxy)-4-oxohutanoic acid (Compound3.2)

To a solution of 4-(tert-butoxycarbonyl(methyl)aminooxy)-4-oxobutanoicacid (96 mg, 0.389) in dioxane (3 mL) was added 4 M HCl solution indioxane (2 mL). The reaction mixture was stirred at room temperature for20 h and concentrated. The desired product was obtained as a white solid(72 mg, 0.389 mmol, 100%). ¹H NMR. (CD₃OD, 400 MHz): 8 (ppm) 2.53-2.57(m, 2H), 2.68-2.77 (m, 2H), 2.94 (s, 3H).

Example 30 Synthesis of αGal epitopeGalα1-3Galα1-4Glc-L₁-N-hydroxysuccinimide ester (NLG αGal-004)

All the linkers proposed so far are short linker where the sugar moietyand peptide are separated by 4-8 carbon/oxygen atoms. To facilitateester cleavage by intracellular esterases and solubility of the linkerfragment we increased the spacing to 12 carbon/oxygen atoms. The newlinker 4.4 would be synthesized according to Scheme 4 shown in FIG. 6.Treatment of N-methyl-N-boc hydroxylamine with 2-(2-chloroethoxy)ethanol gave primary alcohol 4.1 in 72% yield. The alcohol was coupledwith monomethyl adipate in presence of DCC to produce ester 4.2 in 62%yield. Hydrolysis of methyl ester followed by deprotection of boc wouldresult in linker 4.4. This linker would be coupled with αGaltrisaccharide and then activated with N-hydroxy succinimide (NHS) toyield NLG-αGal-004.

Example 31 Synthesis of tert-butyl2-(3-hydroxypropoxy)ethoxy(methyl)carbamate (Compound 4.1)

A mixture of N-Boc-N-methyl hydroxylamine (132 mg, 0.898 mmol),2-(2-chloroethoxy) ethanol (168 mg, 1.35 mmol), K₂CO₃ (372 mg, 2.69mmol) and LiBr (2 mg, 0.018 mmol) in DMF (3 mL) was heated at 100° C.and stirred for 16 h. The resulting mixture was cooled down to roomtemperature and filtered. The filtrate was concentrated and the residuewas purified by silica gel flash column chromatography using 50%EtOAc/hexanes as eluent to afford the desired product as colorless oil(152 mg, 0.647 mmol, 72%). ¹H NMR (CDCl₃, 400 MHz): 8 (ppm) 1.45 (s,9H), 2.68 (br s, 1H), 3.08 (s, 3H), 3.57-3.60 (m, 2H), 3.66-3.72 (m,4H), 3.96-3.99 (m, 2H).

Example 32 Synthesis of methyl2,2,5-trimethyl-4-oxo-3,6,9-trioxa-5-azaundecan-11-yl adipate (Compound4.2)

To a solution of tert-butyl 2-(3-hydroxypropoxy)ethoxy(methyl)carbamate(152 mg, 0.646 mmol) and monomethyl adipate (104 mg, 0.646 mmol) inCH₂Cl₂ (4 mL) were added DCC (146 mg, 0.711 mmol) and DMAP (16 mg, 0.129mmol). The reaction mixture was stirred at room temperature for 16 h andfiltered. The solvent was removed under reduced pressure. The crudeproduct was purified by silica gel flash column chromatography using 27%EtOAc/hexanes as eluent. The desired product was obtained as colorlessoil (152 mg, 0.403 mmol, 62%). ¹H NMR (CDCl₃, 400 MHz): 6 (ppm) 1.41 (s,9H), 1.56-1.60 (m, 4H), 2.25-2.27 (m, 4H), 3.03 (s, 3H), 3.58 (s, 3H),3.59-3.63 (m, 4H), 3.91-3.94 (m, 2H), 4.14-4.16 (m, 2H).

Example 33 Synthesis of4-[(β-D-lactopyranosyl)(methyl)aminooxy]-4-oxobutanoic acid

The methods described for the synthesis of activated αGal epitopes aregenerally applicable to any saccharide. As an additional example, theactivation of lactose with linker of formula (II) was performed by thefollowing procedure. A solution of Lactose (140 mg, 0.408 mmol) and4-(methylaminooxy)-4-oxobutanoic acid (30 mg, 0.204) in DMF/AcOH (1:1)was stirred at room temperature for 24 h. After removal of the solventunder reduced pressure, the crude product was purified by silica flashcolumn chromatography using 35% MeOH/EtOAc as eluent. The desiredproduct was obtained as a white solid (54 mg, 0.121 mmol, 57%). ¹H NMR(CD₃OD, 300 MHz): δ (ppm) 2.66 (s, 3H), 2.77-2.86 (m, 4H), 3.40-3.58 (m,12H), 3.70-3.90 (m, 12H), 4.35 (d, 2H, J=7.5 Hz), 4.49 (d, 1H, J=7.8Hz), 5.09 (d, 1H, J=3.6 Hz).

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1-40. (canceled)
 41. A composition of chemical structureS—N(CH₃)—O—R₁—COOR₂, wherein S is a monosaccharide, disaccharide,tetrasaccharide, or pentasaccharide, wherein R₁ is any linear orbranched alkyl group of 1 to 30 carbon atoms, wherein one or more ofsaid carbon atoms in said alkyl group can be substituted by O, S, or Nand wherein one or more hydrogens can be substituted by hydroxylcarbonyl, alkyl, sulphydryl, or amino groups, and wherein R₂ is an aminoor sulphydryl reacting group.
 42. The composition of claim 41, whereinR₂ is succinimide or maleimide.
 43. The composition of claim 41, whereinsubstitutions of the R₁ alkyl chain create an ester group. 44-49.(canceled)